Pd-1 antibodies in combination with a cytokine-secreting cell and methods of use thereof

ABSTRACT

The present invention relates to a method of enhancing the anti-tumor response in a mammal. More particularly, the invention is concerned with combinations comprising a cytokine-secreting cell and an anti-PD-1 antibody, and methods of administering the combination for enhanced immune response to tumor cells in a patient with a cancer.

This application is a continuation of U.S. patent application Ser. No.14/078,042, filed Nov. 12, 2013, which is a continuation of U.S. patentapplication Ser. No. 13/619,699, filed Sep. 14, 2012, now U.S. Pat. No.8,580,247, issued Nov. 12, 2013, which is a divisional of U.S. patentapplication Ser. No. 12/839,163, filed Jul. 19, 2010, now U.S. Pat. No.8,287,856, issued Oct. 16, 2012, which is a continuation of U.S. patentapplication Ser. No. 12/178,122, filed Jul. 23, 2008, now abandoned,which claims benefit of priority of U.S. provisional Application No.60/961,743, filed Jul. 23, 2007, the contents of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for enhancinganti-tumor protection in a mammal. More particularly, the invention isconcerned with combinations comprising an antibody that specificallybinds to human Programmed Death 1 (PD-1) and a cytokine-secreting cell,and methods of administering the combination for enhanced generation ofan immune response to tumor cells in a patient.

BACKGROUND OF THE INVENTION

The immune system plays a critical role in the pathogenesis of a widevariety of cancers. When cancers progress, it is widely believed thatthe immune system either fails to respond sufficiently or fails torespond appropriately, allowing cancer cells to grow. Currently,standard medical treatments for cancer, including chemotherapy, surgery,radiation therapy and cellular therapy, have clear limitations withregard to both efficacy and toxicity. To date, these approaches have metwith varying degrees of success dependent upon the type of cancer,general health of the patient, stage of disease at the time ofdiagnosis, etc. Improved strategies that combine specific manipulationof the immune response to cancer in combination with standard medicaltreatments may provide a means for enhanced efficacy and decreasedtoxicity.

The use of autologous cancer cells as immunotherapies to augmentanti-tumor immunity has been explored for some time (Oettgen et al.,“The History of Cancer Immunotherapy”, In: Biologic Therapy of Cancer,Devita et al. (eds.) J. Lippincot Co., pp 87-199, 1991). However, due tothe weak immunogenicity of many cancers, down regulation of MHCmolecules, the lack of adequate costimulatory molecule expression andsecretion of immunoinhibitory cytokines by cancer cells, the response tosuch immunotherapies has not resulted in long term efficacy. See, e.g.,Armstrong T D and Jaffee E M, Surg Oncol Clin N Am. 11(3):681-96, 2002and Bodey B et al, Anticancer Res 20(4):2665-76, 2000.

Numerous cytokines have been shown to play a role in regulation of theimmune response to tumors. For example, U.S. Pat. No. 5,098,702describes using combinations of TNF, IL-2 and IFN-beta insynergistically effective amounts to combat existing tumors. U.S. Pat.Nos. 5,078,996, 5,637,483 and 5,904,920 describe the use of GM-CSF fortreatment of tumors. However, direct administration of cytokines forcancer therapy may not be practical, as they are often systemicallytoxic. (See, for example, Asher et al., J. Immunol. 146: 3227-3234, 1991and Havell et al, J. Exp. Med. 167: 1067-1085, 1988.)

An expansion of this approach involves the use of genetically modifiedtumor cells which express cytokines locally at the immunotherapy site.Activity has been demonstrated in tumor models using a variety ofimmunomodulatory cytokines, including IL-4, IL-2, TNF-alpha, G-CSF,IL-7, IL-6 and GM-CSF, as described in Golumbeck P T et al., Science254:13-716, 1991; Gansbacher B et al, J. Exp. Med. 172:1217-1224, 1990;Fearon E R et al., Cell 60:397-403, 1990; Gansbacher B et al., CancerRes. 50:7820-25, 1990; Teng M et al, PNAS 88:3535-3539, 1991; Columbo MPetal, J. Exp. Med. 174:1291-1298, 1991; Aoki et al., Proc Natl Acad SciUSA. 89(9):3850-4, 1992; Porgador A, et al., Nat Immun. 13(2-3):113-30,1994; Dranoff G et al., PNAS 90:3539-3543, 1993; Lee C T et al, HumanGene Therapy 8:187-193, 1997; Nagai E et al, Cancer Immunol. Immonther.47:2-80, 1998 and Chang A et al., Human Gene Therapy 11:839-850, 2000,respectively.

Clinical trials employing GM-CSF-expressing autologous or allogeneiccellular immunotherapies have commenced for treatment of prostatecancer, melanoma, lung cancer, pancreatic cancer, renal cancer, andmultiple myeloma (Dummer R., Curr Opin Investig Drugs 2(6):844-8, 2001;Simons J et al, Cancer Res. 15; 59(20):5160-8, 1999; Soiffer R et al.,PNAS 95:13141-13146, 1998; Simons J et al., Cancer Res. 15;57:1537-1546,1997; Jaffee E et al, J. Clin Oncol. 19:145-156, 2001; andSalgia R et al, J. Clin Oncol. 21:624-630, 2003).

In yet another approach, autologous tumor cells were genetically alteredto produce a costimulatory molecule, such as B7-1 or allogeneichistocompatibility antigens (Salvadori et al. Hum. Gene Ther.6:1299-1306, 1995 and Plaksin et al. Int. J. Cancer 59:796-801, 1994).While the use of genetically modified tumor cells has met with successin treatment of some forms of cancer, there remains a need for improvedtreatment regimens with greater potency and/or efficacy and fewer sideeffects than the therapies currently in use.

SUMMARY OF THE INVENTION

The invention provides improved compositions and methods for thetreatment of cancer in a mammal, typically a human, by administering acombination of a cytokine-expressing cellular immunotherapy and anantibody that specifically binds to human Programmed Death (PD)-1.

In one aspect of the invention, the cytokine-expressing cellularimmunotherapy expresses GM-CSF.

In another aspect of the invention, the cytokine-expressing cellularimmunotherapy is rendered proliferation-incompetent by irradiation.

In yet a further aspect of the invention, administration of thecombination results in enhanced therapeutic efficacy relative toadministration of the cytokine-expressing cellular immunotherapy or theanti-PD-1 antibody alone.

In yet another aspect of the invention, the cytokine-expressing cellularimmunotherapy is typically administered subcutaneously, intratumorally,or intradermally. The injection of irradiated GM-CSF-expressing tumorcells results in a local reaction characterized by the infiltration ofdendritic cells (DCs), macrophages, and granulocytes.

In another aspect of the invention, the anti-PD-1 antibody may beadministered prior to, at the same time as, or following administrationof the cytokine-expressing cellular immunotherapy component of thecombination. The anti-PD-1 antibody may be administered via parenteral,e.g., subcutaneous, intratumoral, intravenous, intradermal, oral,transmucosal, or rectal administration. While not intending to be boundto a particular theory of operation, it is believed that blockade ofPD-1 through the administration of an anti-PD-1 antibody potentiatesanti-tumor immunity by negatively modulating the immunoinhibitoryeffects of PD-1 signaling in activated T-cells, B-cells and myeloidcells.

The invention further provides a combination of cytokine-expressingcells and an anti-PD-1 antibody, wherein the combination comprises cellsthat are autologous, allogeneic, or bystander cells.

In another aspect of the invention, the autologous, allogeneic, orbystander cell is rendered proliferation-incompetent by irradiation.

The invention further provides compositions and kits comprisingcytokine-expressing cellular immunotherapy combinations for useaccording to the description provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the anti-tumor T-cell response in an adoptivetransfer model in C57BL/6 mice challenged with live B16F10 tumor cellsmodified to express ovalbumin as a surrogate antigen (B16.ova) andimmunized with GM-CSF-secreting B16.ova cells (GM.ova) alone, or incombination with an anti-PD-1 antibody. The number of ovalbumin-specificT-cells in draining lymph nodes (DLN) (top panel) and spleens (bottompanel) were determined by tetramer staining at 3, 7, 10, 14 and 20 daysfollowing immunotherapy.

FIG. 1B illustrates the cytolytic activity of T-cells in mice challengedwith live B16 tumor cells modified to express ovalbumin as a surrogateantigen (B16.ova) and immunized with GM-CSF secreting B16.ova cells(GM.ova) alone, or in combination with an anti-PD-1 antibody. Cytolyticactivity was determined by assessing the ratio of CSFE-labeled cells inisolated splenocytes following injection of CSFE-labeled non-pulsed andSIINFEKL peptide pulsed syngeneic splenocytes. Cytolytic activity wasassessed at 7, 14, 21 and 28 days post immunotherapy.

FIG. 1C illustrates the number of IFNγ-secreting cells per 5×10⁵splenocytes isolated from mice challenged with live B16 tumor cells,then immunized with allogeneic GM-CSF-secreting B16F10 cells alone(B16.Kd.GM), or in combination with an anti-PD-1 antibody. Controlanimals were administered HBSS at the time of immunization. Isolatedsplenocytes were stimulated with trp2 peptide (top panel) or irradiatedB16F10 cells (bottom panel) before being assayed by ELISPOT.

FIG. 2A illustrates the secretion of the pro-inflammatory cytokine tumornecrosis factor-alpha (TNFα) from splenocytes isolated from micechallenged with live tumor cells, then immunized with GM-CSF-secretingB16F10 cells alone (B16.Kd.GM), or in combination with an anti-PD-1antibody. Splenocytes were stimulated with irradiated B16 cells for 48hours before assaying for cytokine secretion.

FIG. 2B illustrates the secretion of the pro-inflammatory cytokineinterferon-gamma (IFNγ) from splenocytes isolated from mice challengedwith live tumor cells, then immunized with GM-CSF-secreting B16F10 cellsalone (B16.Kd.GM), or in combination with an anti-PD-1 antibody.Splenocytes were stimulated with irradiated B16 cells for 48 hoursbefore assaying for cytokine secretion.

FIG. 2C illustrates the secretion of the pro-inflammatory cytokineinterleukin-5 (IL-5) from splenocytes isolated from mice challenged withlive tumor cells, then immunized with GM-CSF-secreting B16F10 cellsalone (B16.Kd.GM), or in combination with an anti-PD-1 antibody.Splenocytes were stimulated with irradiated B16 cells for 48 hoursbefore assaying for cytokine secretion.

FIG. 2D illustrates the secretion of the pro-inflammatory cytokineinterleukin-6 (IL-6) from splenocytes isolated from mice challenged withlive tumor cells, then immunized with GM-CSF-secreting B16F10 cellsalone (B16.Kd.GM), or in combination with an anti-PD-1 antibody.Splenocytes were stimulated with irradiated B16 cells for 48 hoursbefore assaying for cytokine secretion.

FIG. 2E illustrates the secretion of the pro-inflammatory cytokineinterleukin-10 (IL-10) from splenocytes isolated from mice challengedwith live tumor cells, then immunized with GM-CSF-secreting B16F10 cellsalone (B16.Kd.GM), or in combination with an anti-PD-1 antibody.Splenocytes were stimulated with irradiated B16 cells for 48 hoursbefore assaying for cytokine secretion.

FIG. 2F illustrates the secretion of the pro-inflammatory cytokinemonocyte chemotactic protein (MCP)-1 from splenocytes isolated from micechallenged with live tumor cells, then immunized with GM-CSF-secretingB16F10 cells alone (B16.Kd.GM), or in combination with an anti-PD-1antibody. Splenocytes were stimulated with irradiated B16 cells for 48hours before assaying for cytokine secretion.

FIG. 3A illustrates effector CD8 T-cell infiltration into tumors of micechallenged with live tumor cells, then immunized with GM-CSF-secretingB16F10 cells alone (B16.Kd.GM) or with GM-CSF-secreting B16F10 cells incombination with an anti-PD-1 antibody (combo). Shown are the percentageof IFNγ and CD107α expressing cells in the CD8 TIL subpopulation.

FIG. 3B illustrates effector CD8 T-cell infiltration into tumors of micechallenged with live tumor cells, then immunized with GM-CSF-secretingB16F10 cells alone (B16.Kd.GM) or with GM-CSF-secreting B16F10 cells incombination with an anti-PD-1 antibody. Shown is the ratio ofCD8+/FoxP3+ cells in the tumor at 3 wks post cellular immunotherapy.

FIG. 3C illustrates the kinetics of CD4⁺ T-cell infiltration into tumorcells (as measured per 1×10⁶ tumor cells) in mice challenged with livetumor cells, then immunized with GM-CSF-secreting B16F10 cells alone(B16.Kd.GM), or in combination with an anti-PD-1 antibody. CD4⁺ T-cellcounts were determined at 7, 14 and 21 days post cellular immunotherapy.

FIG. 3D illustrates the kinetics of CD8⁺ T-cell infiltration into tumorcells (as measured per 1×10⁶ tumor cells) in mice challenged with livetumor cells, then immunized with GM-CSF-secreting B16F10 cells alone(B16.Kd.GM), or in combination with an anti-PD-1 antibody. CD8⁺ T-cellcounts were determined at 7, 14 and 21 days post cellular immunotherapy.

FIG. 3E illustrates the kinetics of CD8⁺/107a⁺ T-cell infiltration intotumor cells (as measured per 1×10⁶ tumor cells) in mice challenged withlive tumor cells, then immunized with GM-CSF-secreting B16F10 cellsalone (B16.Kd.GM), or in combination with an anti-PD-1 antibody.CD8⁺/107a⁺ T-cell counts were determined at 7, 14 and 21 days postcellular immunotherapy.

FIG. 3F illustrates the kinetics of CD8⁺/IFNγ⁺ T-cell infiltration intotumor cells (as measured per 1×10⁶ tumor cells) in mice challenged withlive tumor cells, then immunized with GM-CSF-secreting B16F10 cellsalone (B16.Kd.GM), or in combination with an anti-PD-1 antibody.CD8⁺/IFNγ⁺ T-cell counts were determined at 7, 14 and 21 days postcellular immunotherapy.

FIG. 3G illustrates tumor progression in mice challenged with live tumorcells, then immunized with GM-CSF-secreting B16F10 cells alone(B16.Kd.GM), or in combination with an anti-PD-1 antibody. Tumors frommice (n=5/group) were excised, digested and single cell suspensions fromthe entire digest were counted by cell counter. Counts were determinedat 7, 14 and 21 days post cellular immunotherapy.

FIG. 4A illustrates the survival of B16F10 tumor-bearing animalsfollowing administration of GM-CSF-secreting B16F10 cells alone(B16.Kd.GM) or in combination with an anti-PD-1 antibody, when given onday 3 post tumor challenge.

FIG. 4B illustrates the survival of B16F10 tumor-bearing animalsfollowing administration of GM-CSF-secreting B16F10 cells alone(B16.Kd.GM) or in combination with an anti-PD-1 antibody, when given onday 7 post tumor challenge.

FIG. 4C illustrates the survival of B16F10 tumor-bearing animalsfollowing administration of GM-CSF-secreting cells alone (B16.Kd.GM) orin combination with an anti-PD-1 antibody, when given on day 11 posttumor challenge.

FIG. 4D illustrates the memory response of mice which survived aninitial tumor challenge following administration of GM-CSF-secretingcells alone or in combination with an anti-PD-1 antibody. On day 90after initial tumor challenge, animals were re-challenged with 5×10⁵(2.5 fold of initial dose) B16 tumor cells and monitored for survival. AKaplan-Meier survival curve was used for evaluation.

FIG. 5A illustrates the survival of CT26 tumor-bearing animals followingadministration of GM-CSF-secreting CT26 cells alone (CT26.GM), anti-PD-1antibody alone (anti-PD-1) or CT26.GM in combination with an anti-PD-1antibody when given on day 3 post tumor challenge.

FIG. 5B illustrates the tumor burden of animals following administrationof GM-CSF-secreting CT26 cells alone (CT26.GM), anti-PD-1 antibody alone(anti-PD-1) or CT26.GM in combination with an anti-PD-1 antibody whengiven on day 3 post tumor challenge.

FIG. 6A illustrates the percentage of CD4⁺ T-cells in the spleens ofmice treated with GM-CSF-secreting B16F10 cells alone (B16.Kd.GM) or incombination with an anti-PD-1 antibody.

FIG. 6B illustrates the percentage of CD8⁺ T-cells in the spleens ofmice treated with GM-CSF-secreting B16F10 cells alone (B16.Kd.GM) or incombination with an anti-PD-1 antibody.

FIG. 6C illustrates the percentage of CD11c⁺ T-cells in the spleens ofmice treated with GM-CSF-secreting B16F10 cells alone (B16.Kd.GM) or incombination with an anti-PD-1 antibody.

FIG. 6D illustrates the percentage of DX5⁺ T-cells in the spleens ofmice treated with GM-CSF-secreting B16F10 cells alone (B16.Kd.GM) or incombination with an anti-PD-1 antibody.

FIG. 7A illustrates the percentage of memory T-cells (Ly6C⁺/CD69⁻) inthe CD4 subpopulation from spleen taken from mice treated withGM-CSF-secreting B16F10 cells alone (B16.Kd.GM) or in combination withan anti-PD-1 antibody.

FIG. 7B illustrates the percentage of memory T-cells (Ly6C⁺/CD69⁻) inthe CD8 subpopulation from spleen taken from mice treated withGM-CSF-secreting B16F10 cells alone (B16.Kd.GM) or in combination withan anti-PD-1 antibody.

FIG. 8 illustrates the reversal of anergy and augmentation of atumor-specific T-cell response in C57BL/6 mice injected with SIINFEKLpeptide (to induce anergy in adoptively transferred OT-1 transgenicT-cells) and immunized with GM-CSF-secreting B16.ova cells (GM.ova)alone, or in combination with an anti-PD-1 antibody. The percentage ofantigen-specific T-cells in peripheral blood lymphocytes was determinedby tetramer staining at 4, 10, 14, 17, 25 and 31 days following adoptivetransfer.

FIG. 9(A)-(C) illustrates the expansion and contraction of thepopulation of ovalbumin-specific CD8 T-cells in spleens of mice treatedwith an initial therapy cycle of GM.ova immunotherapy, followed byeither (A) anti-PD-1 and immunotherapy administered biweekly; (B) GM.ovaimmunotherapy only administered biweekly; or (V) anti-PD-1 administeredbiweekly and GM.ova immunotherapy administered monthly. Shown are theabsolute numbers of ovalbumin-specific CD8 T-cells in the spleen bytetramer staining.

FIG. 9(D)-(F) illustrates the expansion and contraction of thepopulation of ovalbumin-specific CD8 T-cells in spleens of mice treatedwith an initial therapy cycle of GM.ova immunotherapy, followed byeither (D) anti-PD-1 and immunotherapy administered biweekly; (E) GM.ovaimmunotherapy only administered biweekly; or (F) anti-PD-1 administeredbiweekly and GM.ova immunotherapy administered monthly. Shown are theabsolute numbers of ovalbumin-specific CD8 T-cells co-stained with theactivation marker CD107a.

FIG. 9(G)-(I) illustrates the expansion and contraction of thepopulation of ovalbumin-specific CD8 T-cells in spleens of mice treatedwith an initial therapy cycle of GM.ova immunotherapy, followed byeither (G) anti-PD-1 and immunotherapy administered biweekly; (H) GM.ovaimmunotherapy only administered biweekly; or (I) anti-PD-1 administeredbiweekly and GM.ova immunotherapy administered monthly. Shown are theabsolute numbers of ovalbumin-specific CD8 T-cells co-stained with IFN-γ(C).

DETAILED DESCRIPTION OF THE INVENTION

The present invention represents improved cellular immunotherapies forthe treatment of cancer in that the compositions and methods describedherein comprise at least two components that act in concert to effect animproved therapeutic outcome for the cancer patient under treatment.

The invention is not limited to the specific compositions andmethodology described herein. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention.

DEFINITIONS

The terms “regulating the immune response” or “modulating the immuneresponse” as used herein refers to any alteration in a cell of theimmune system or any alteration in the activity of a cell involved inthe immune response. Such regulation or modulation includes an increaseor decrease in the number of various cell types, an increase or decreasein the activity of these cells, or any other changes which can occurwithin the immune system. Cells involved in the immune response include,but are not limited to, T lymphocytes, B lymphocytes, natural killer(NK) cells, macrophages, eosinophils, mast cells, dendritic cells andneutrophils. In some cases, “regulating” or “modulating” the immuneresponse means the immune response is stimulated or enhanced, and inother cases “regulating” or “modulating” the immune response meanssuppression of the immune system. Stimulation of the immune system mayinclude memory responses and/or future protection against subsequentantigen challenge.

The term “cytokine” or “cytokines” as used herein refers to the generalclass of biological molecules which effect/affect cells of the immunesystem. The definition is meant to include, but is not limited to, thosebiological molecules that act locally or may circulate in the blood, andwhich, when used in the compositions or methods of the present inventionserve to regulate or modulate an individual's immune response to cancer.Exemplary cytokines for use in practicing the invention include but arenot limited to interferon-alpha (IFN-α), interferon-beta (IFN-β), andinterferon-gamma (IFN-γ), interleukins (e.g., IL-1 to IL-29, inparticular, IL-2, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15 and IL-18),tumor necrosis factors (e.g., TNF-alpha and TNF-beta), erythropoietin(EPO), MIP3a, monocyte chemotactic protein (MCP)-1, intracellularadhesion molecule (ICAM), macrophage colony stimulating factor (M-CSF),granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophagecolony stimulating factor (GM-CSF).

The term “cytokine-expressing cellular immunotherapy” as used hereinrefers to a composition comprising a population of cells that has beengenetically modified to express a cytokine, e.g., GM-CSF, and that isadministered to a patient as part of a cancer treatment regimen. Thecells of such a “cytokine-expressing cellular immunotherapy” comprise acytokine-encoding DNA sequence operably linked to expression and controlelements such that the cytokine is expressed by the cells. The cells ofthe “cytokine-expressing cellular immunotherapy” are typically tumorcells and may be autologous or allogeneic to the patient undergoingtreatment and or may be “bystander cells” that are mixed with tumorcells taken from the patient. A GM-CSF-expressing “cytokine-expressingcellular immunotherapy” may be referred to herein as “GVAX®”.

The term “operably linked” as used herein relative to a recombinant DNAconstruct or vector means nucleotide components of the recombinant DNAconstruct or vector are directly linked to one another for operativecontrol of a selected coding sequence. Generally, “operably linked” DNAsequences are contiguous, and, in the case of a secretory leader,contiguous and in reading frame; however, some sequences, e.g.,enhancers do not have to be contiguous to be operative and therefore“operably linked.”

As used herein, the term “gene” or “coding sequence” means the nucleicacid sequence which is transcribed (DNA) and translated (mRNA) into apolypeptide in vitro or in vivo when operably linked to appropriateregulatory sequences. A “gene” typically comprises the coding sequenceplus any non-coding sequences associated with the gene (e.g., regulatorysequences) and hence may or may not include regions preceding andfollowing the coding region, e.g., 5′ untranslated (5′UTR) or “leader”sequences and 3′ UTR or “trailer” sequences, as well as interveningsequences (introns) between individual coding segments (exons). Incontrast, a “coding sequence” does not include non-coding DNA.

The terms “gene-modified” and “genetically-modified” are used hereinwith reference to a cell or population of cells wherein a nucleic acidsequence has been introduced into the cell or population of cells. Thenucleic acid sequence may be heterologous to the cell(s), or it may bean additional copy or altered version of a nucleic acid sequence alreadypresent in the cell(s). This term also encompasses cells or a populationof cells with altered, e.g., increased or decreased, expression of anucleic acid sequence endogenous to the cell or population of cells. Thecell(s) may be genetically-modified by physical or chemical methods orby the use of recombinant viruses. Chemical and physical methods such ascalcium phosphate, electroporation and pressure mediated transfer ofgenetic material into cells are often used. Several recombinant viralvectors which find utility in effective delivery of genes into mammaliancells include, for example, retroviral vectors, adenovirus vectors,adenovirus-associated vectors (AAV), herpes virus vectors, pox virusvectors. In addition, non-viral means of introduction, for example,naked DNA delivered via liposomes, receptor-mediated delivery, calciumphosphate transfection, electroporation, particle bombardment (genegun), or pressure-mediated delivery may also be employed to introduce anucleic acid sequence into a cell or population of cells to render them“gene-modified” or “genetically-modified.

The terms “cancer,” “cancer cells,” “neoplastic cells,” “neoplasia,”“tumor,” and “tumor cells” (used interchangeably) refer to cells thatexhibit relatively autonomous growth, so that they exhibit an aberrantgrowth phenotype or aberrant cell status characterized by a significantloss of control of cell proliferation. A tumor cell may be ahyperplastic cell, a cell that shows a lack of contact inhibition ofgrowth in vitro or in vivo, a cell that is incapable of metastasis invivo, or a cell that is capable of metastasis in vivo. Neoplastic cellscan be malignant or benign. It follows that cancer cells are consideredto have an aberrant cell status.

The term “antigen from a tumor cell” and “tumor antigen” and “tumor cellantigen” may be used interchangeably herein and refer to any protein,carbohydrate or other component derived from or expressed by a tumorcell which is capable of eliciting an immune response. The definition ismeant to include, but is not limited to, whole tumor cells that expresssome or all of the tumor-associated antigens, tumor cell fragments,plasma membranes taken from a tumor cell, proteins purified from thecell surface or membrane of a tumor cell, or unique carbohydratemoieties associated with the cell surface of a tumor cell. Thedefinition also includes those antigens from the surface of the cellwhich require special treatment of the cells to access.

As described herein, a “tumor cell line” comprises cells that wereinitially derived from a tumor. Such cells typically are transformed(i.e., exhibit indefinite growth in culture).

The term “systemic immune response” as used herein means an immuneresponse which is not localized, but affects the individual as a whole.

The term “gene therapy” as used herein means the treatment or preventionof cancer by means of ex vivo or in vivo delivery, through viral ornon-viral vectors, of compositions containing a recombinant geneticmaterial.

The term “ex vivo” delivery as used herein means the introduction,outside of the body of a human, of compositions containing a geneticmaterial into a cell, tissue, organoid, organ, or the like, followed bythe administration of cell, tissue, organoid, organ, or the like whichcontains such introduced compositions into the body of the same(autologous) or a different (allogeneic) human, without limitation as tothe formulation, site or route of administration.

The terms “inactivated cells,” “non-dividing cells” and “non-replicatingcells” may be used interchangeably herein and refer to cells that havebeen treated rendering them proliferation incompetent, e.g., byirradiation. Such treatment results in cells that are unable to undergomitosis, but retain the capability to express proteins such as cytokinesor other cancer therapeutic agents. Typically a minimum dose of about3500 rads is sufficient, although doses up to about 30,000 rads areacceptable. Effective doses include, but are not limited to, 5000 to10000 rads. Numerous methods of inactivating cells, such as treatmentwith Mitomycin C, are known in the art. Any method of inactivation whichrenders cells incapable of cell division, but allows the cells to retainthe ability to express proteins may be used in accordance with thepresent invention.

As used herein “treatment” of an individual or a cell is any type ofintervention used in an attempt to alter the natural course of theindividual or cell. Treatment includes, but is not limited to,administration of e.g., a cytokine-expressing cellular immunotherapy andat least one additional cancer therapeutic agent or treatment, e.g. ananti-PD-1 antibody, and may be performed either prophylactically orsubsequent to diagnosis as part of a primary or follow-up therapeuticregimen.

The term “administering” as used herein refers to the physicalintroduction of a composition comprising a cytokine-expressing cellularimmunotherapy and at least one additional cancer therapeutic agent ortreatment to a patient with cancer. Any and all methods of introductionare contemplated according to the invention; the method is not dependenton any particular means of introduction. Means of introduction arewell-known to those skilled in the art, examples of which are providedherein.

The term “co-administering” as used herein means a process whereby thecombination of a cytokine-expressing cellular immunotherapy and at leastone additional cancer therapeutic agent, e.g., an anti-PD-1 antibody, isadministered to the same patient. The cytokine-expressing cellularimmunotherapy and additional cancer therapeutic may be administeredsimultaneously, at essentially the same time, or sequentially. Ifadministration takes place sequentially, the cytokine-expressingcellular immunotherapy may be administered before or after a givenadditional cancer therapeutic agent or treatment. Thecytokine-expressing cellular immunotherapy and additional cancertherapeutic agent or treatment need not be administered by means of thesame vehicle. The cellular immunotherapy and the additional agent ortreatment may be administered one or more times and the number ofadministrations of each component of the combination may be the same ordifferent. In addition, the cytokine-expressing cellular immunotherapyand additional cancer therapeutic agent or treatment need not beadministered at the same site.

The term “therapeutically effective amount” or “therapeuticallyeffective combination” as used herein refers to an amount or dose of acytokine-expressing cellular immunotherapy together with the amount ordose of an additional agent or treatment, e.g. an anti-PD-1 antibody,that is sufficient to modulate, either by stimulation or suppression,the systemic immune response of an individual. The amount ofcytokine-expressing cellular immunotherapy in a given therapeuticallyeffective combination may be different for different individuals anddifferent tumor types, and will be dependent upon the one or moreadditional agents or treatments included in the combination. The“therapeutically effective amount” is determined using proceduresroutinely employed by those of skill in the art such that an “improvedtherapeutic outcome” results.

As used herein, the terms “improved therapeutic outcome” and “enhancedtherapeutic efficacy,” relative to cancer refers to a slowing ordiminution of the growth of cancer cells or a solid tumor, or areduction in the total number of cancer cells or total tumor burden. An“improved therapeutic outcome” or “enhanced therapeutic efficacy”therefore means there is an improvement in the condition of the patientaccording to any clinically acceptable criteria, including, for example,decreased tumor size, an increase in time to tumor progression,increased progression-free survival, increased overall survival time, anincrease in life expectancy, or an improvement in quality of life. Inparticular, “improved” or “enhanced” refers to an improvement orenhancement of 1%, 5%, 10%, 25% 50%, 75%, 100%, or greater than 100% ofany clinically acceptable indicator of therapeutic outcome or efficacy.

As used herein, the term “synergism,” “synergistic” or “synergistically”refers to the combined action of two or more agents wherein the combinedaction is greater than the sum of the actions of each of the agents usedalone.

The term “relative to” or “compared to,” when used in the context ofcomparing the activity and/or efficacy of a combination compositioncomprising the cytokine-expressing cellular immunotherapy (GVAX) plus ananti-PD-1 antibody (anti-PD-1) to either GVAX or anti-PD-1 alone, refersto a comparison using amounts known to be comparable according to one ofskill in the art. Comparable amounts of GVAX, when comparing thecombination therapy to GVAX alone, may be based on cell number, cytokineexpression, cytokine secretion, or cytokine activity on a per cellbasis. Comparable amounts of anti-PD-1, when comparing the combinationtherapy to PD-1 alone, may be based on equimolar amounts,weight-to-weight equivalents, or units of PD-1 binding activity.

The term “reversal of an established tumor” as used herein means thesuppression, regression, partial or complete disappearance of apre-existing tumor. The definition is meant to include any diminution,for example, in the size, growth rate, appearance or cellularcompositions of a preexisting tumor.

The terms “individual” or “subject” as referred to herein is avertebrate, preferably a mammal, and typically refers to a human.

The terms “programmed death-1,” “programmed death receptor-1” and “PD-1”are synonymous with one another, and include variants, isoforms, specieshomologs of human PD-1, and analogs having at least one common epitopewith PD-1. The full length human PD-1 cDNA is 2106 nucleotides long andencodes a protein of 288 amino acid residues. The human PD-1 and murinePD-1 genes share 70% homology at the nucleotide level and 60% homologyat the amino acid level. The complete cDNA sequence of human PD-1 hasthe Genbank accession number U64863 (Shinohara et al., Genomics 23(3):704-706 (1994). The extracellular domain is encoded by amino acids1-166; the transmembrane domain is encoded by amino acids 167-196; andthe cytoplasmic domain is encoded by amino acids 197-288. Theextracellular domain contains an immunoglobulin superfamily domain, andthe cytoplasmic domain includes an immunoreceptor tyrosine-basedinhibitory motif (ITIM). The complete cDNA sequence of murine PD-1 hasthe Genbank accession number X67914 (Ishida et al., EMBO J.11(11):3887-3895 (1992)).

An intact “antibody” comprises at least two heavy (H) chains and twolight (L) chains inter-connected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as HCVRor VH) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. Each light chainis comprised of a light chain variable region (abbreviated herein asLCVR or VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with PD-1. The constant regions of theantibodies may mediate the binding of the immunoglobulin to host tissuesor factors, including various cells of the immune system (e.g., effectorcells) and the first component (Clq) of the classical complement system.The term antibody includes antigen-binding portions of an intactantibody that retain capacity to bind PD-1. Examples of binding include(i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CLand CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprisingtwo Fab fragments linked by a disulfide bridge at the hinge region;(iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fvfragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); See,e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodiesare included by reference to the term “antibody.” Fragments can beprepared by recombinant techniques or enzymatic or chemical cleavage ofintact antibodies.

“Conservative substitution” refers to the substitution in a polypeptideof an amino acid with a functionally similar amino acid. The followingsix groups each contain amino acids that are conservative substitutionsfor one another:

-   -   Alanine (A), Serine (S), and Threonine (T)    -   Aspartic acid (D) and Glutamic acid (E)    -   Asparagine (N) and Glutamine (Q)    -   Arginine (R) and Lysine (K)    -   Isoleucine (I), Leucine (L), Methionine (M), and Valine (V)    -   Phenylalanine (F), Tyrosine (Y), and Tryptophan (W).

The term “stringent conditions” refers to conditions under which a probewill hybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. “Stringent hybridization”and “stringent hybridization wash conditions” in the context of nucleicacid hybridization experiments such as Southern and northernhybridizations are sequence dependent, and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids can be found in Tijssen, 1993, Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, part I, chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays”, Elsevier, N.Y.; Sambrook etal., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, 3^(rd) ed., NY; and Ausubel et al., eds., Current Edition,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, NY.

Generally, highly stringent hybridization and wash conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Very stringentconditions are selected to be equal to the Tm for a particular probe.

One example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than about 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of highly stringent wash conditions is 0.15 M NaClat 72° C. for about 15 minutes. An example of stringent wash conditionsis a 0.2×SSC wash at 65° C. for 15 minutes. See Sambrook et al. for adescription of SSC buffer. A high stringency wash can be preceded by alow stringency wash to remove background probe signal. An exemplarymedium stringency wash for a duplex of, e.g., more than about 100nucleotides, is 1×SSC at 45° C. for 15 minutes. An exemplary lowstringency wash for a duplex of, e.g., more than about 100 nucleotides,is 4-6×SSC at 40° C. for 15 minutes. In general, a signal to noise ratioof 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization.

The term “% sequence identity” is used interchangeably herein with theterm “% identity” and refers to the level of amino acid sequenceidentity between two or more peptide sequences or the level ofnucleotide sequence identity between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% identity means the same thing as 80% sequence identitydetermined by a defined algorithm, and means that a given sequence is atleast 80% identical to another length of another sequence. Exemplarylevels of sequence identity include, but are not limited to, 60, 70, 80,85, 90, 95, 98% or more sequence identity to a given sequence.

The term “% sequence homology” is used interchangeably herein with theterm “% homology” and refers to the level of amino acid sequencehomology between two or more peptide sequences or the level ofnucleotide sequence homology between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% homology means the same thing as 80% sequence homologydetermined by a defined algorithm, and accordingly a homologue of agiven sequence has greater than 80% sequence homology over a length ofthe given sequence. Exemplary levels of sequence homology include, butare not limited to, 60, 70, 80, 85, 90, 95, 98% or more sequencehomology to a given sequence.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet at the NCBI website. See alsoAltschul et al., 1990, J. Mol. Biol. 215:403-10 (with special referenceto the published default setting, i.e., parameters w=4, t=17) andAltschul et al., 1997, Nucleic Acids Res., 25:3389-3402. Sequencesearches are typically carried out using the BLASTP program whenevaluating a given amino acid sequence relative to amino acid sequencesin the GenBank Protein Sequences and other public databases. The BLASTXprogram is preferred for searching nucleic acid sequences that have beentranslated in all reading frames against amino acid sequences in theGenBank Protein Sequences and other public databases. Both BLASTP andBLASTX are run using default parameters of an open gap penalty of 11.0,and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix.See id.

Another alignment of selected sequences in order to determine “%identity” between two or more sequences, is performed using for example,the CLUSTAL-W program in MacVector version 6.5, operated with defaultparameters, including an open gap penalty of 10.0, an extended gappenalty of 0.1, and a BLOSUM 30 similarity matrix.

The term “about,” as used herein, unless otherwise indicated, refers toa value that is no more than 10% above or below the value being modifiedby the term. For example, the term “about 5 μg/kg” means a range of from4.5 μg/kg to 5.5 μg/kg. As another example, “about 1 hour” means a rangeof from 54 minutes to 66 minutes.

General Techniques

The practice of the present invention can employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,cell biology, biochemistry and immunology, which are within theknowledge of those of skill of the art. Such techniques are explainedfully in the literature, such as, “Molecular Cloning: A LaboratoryManual”, third edition (Sambrook et al., 2002); “Current Protocols inMolecular Biology” (F. M. Ausubel et al., eds., 2007); “Culture ofAnimal Cells: A Manual of Basic Techniqaue,” 4^(th) edition (R. I.Freshney, ed., 2000), each of which is hereby expressly incorporatedherein by reference.

Cancer Targets

The methods and compositions of the invention provide an improvedtherapeutic approach to the treatment of cancer by co-administration ofa cytokine-expressing cellular immunotherapy and an antibody thatspecifically binds to human PD-1 to a patient with cancer. “Cancer” asused herein includes cancer localized in tumors, as well as cancer notlocalized in tumors, such as, for instance, cancer cells that expandfrom a local tumor by invasion (i.e., metastasis). The invention findsutility in the treatment of any form of cancer, including, but notlimited to, cancer of the bladder, breast, colon, kidney, liver, lung,ovary, cervix, pancreas, rectum, prostate, stomach, epidermis; ahematopoietic tumor of lymphoid or myeloid lineage; a tumor ofmesenchymal origin such as a fibrosarcoma or rhabdomyosarcoma; othertumor types such as melanoma, teratocarci-noma, neuroblastoma, glioma,adenocarcinoma and non-small lung cell carcinoma.

Introduction of Cytokine into Cells

In one aspect of the invention, a nucleic acid sequence (i.e., arecombinant DNA construct or vector) encoding a cytokine operably linkedto a promoter is introduced into a cell or population of cells. Any andall methods of introduction into a cell or population of cells,typically tumor cells, are contemplated according to the invention. Themethod is not dependent on any particular means of introduction and isnot to be so construed.

The “vector” may be a DNA molecule such as a plasmid, virus or othervehicle, which contains one or more heterologous or recombinant DNAsequences, e.g., a nucleic acid sequence encoding a cytokine under thecontrol of a functional promoter and in some cases further including anenhancer that is capable of functioning as a vector, as understood bythose of ordinary skill in the art. An appropriate viral vectorincludes, but is not limited to, a retrovirus, a lentivirus, anadenovirus (AV), an adeno-associated virus (AAV), a simian virus 40(SV-40), a bovine papilloma virus, an Epstein-Ban virus, a herpes virus,a vaccinia virus, a Moloney murine leukemia virus, a Harvey murinesarcoma virus, a murine mammary tumor virus, and a Rous sarcoma virus.Non-viral vectors are also included within the scope of the invention.

Any suitable vector can be employed that is appropriate for introductionof nucleic acids into eukaryotic tumor cells, or more particularlyanimal tumor cells, such as mammalian, e.g., human, tumor cells.Preferably the vector is compatible with the tumor cell, e.g., iscapable of imparting expression of the coding sequence for a cytokineand is stably maintained or relatively stably maintained in the tumorcell. Desirably the vector comprises an origin of replication and thevector may or may not also comprise a “marker” or “selectable marker”function by which the vector can be identified and selected. While anyselectable marker can be used, selectable markers for use in suchexpression vectors are generally known in the art and the choice of theproper selectable marker will depend on the host cell. Examples ofselectable marker genes which encode proteins that confer resistance toantibiotics or other toxins include ampiciilin, methotrexate,tetracycline, neomycin (Southern and Berg, J., 1982), myco-phenolic acid(Mulligan and Berg, 1980), puromycin, zeo-mycin, hygromycin (Sugden etal., 1985) or G418.

In practicing the methods of the present invention, a vector comprisinga nucleic acid sequence encoding a cytokine may be transferred to a cellin vitro, preferably a tumor cell, using any of a number of methodswhich include but are not limited to electroporation, membrane fusionwith liposomes, Lipofectamine treatment, high velocity bombardment withDNA-coated microprojectiles, incubation with calcium phosphate-DNAprecipitate, DEAE-dextran mediated transfection, infection with modifiedviral nucleic acids, direct microinjection into single cells, etc.Procedures for the cloning and expression of modified forms of a nativeprotein using recombinant DNA technology are generally known in the art,as described in Ausubel, et al., 2007 and Sambrook, et al., 2002,expressly incorporated by reference, herein.

Reference to a vector or other DNA sequences as “recombinant” merelyacknowledges the operable linkage of DNA sequences which are nottypically operably linked as isolated from or found in nature. A“promoter” is a DNA sequence that directs the binding of RNA polymeraseand thereby promotes RNA synthesis. “Enhancers” are cis-acting elementsthat stimulate or inhibit transcription of adjacent genes. An enhancerthat inhibits transcription also is termed a “silencer.” Enhancers canfunction (i.e. be operably linked to a coding sequence) in eitherorientation, over distances of up to several kilobase pairs (kb) fromthe coding sequence and from a position downstream of a transcribedregion. Regulatory (expression/control) sequences are operatively linkedto a nucleic acid coding sequence when the expression/control sequencesregulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus, expression/control sequences can includepromoters, enhancers, transcription terminators, a start codon (i.e.,ATG) in front of the coding sequence, a splicing signal for introns, andstop codons.

Recombinant vectors for the production of cellular immunotherapies ofthe invention provide the proper transcription, translation andprocessing signals (e.g., splicing and polyadenylation signals) suchthat the coding sequence for the cytokine is appropriately transcribedand translated in the tumor cells into which the vector is introduced.The manipulation of such signals to ensure appropriate expression inhost cells is within the skill of the ordinary skilled artisan. Thecoding sequence for the cytokine may be under control of (i.e., operablylinked to) its own native promoter, or a non-native (e.g. heterologous)promoter, including a constitutive promoter, e.g., the cytomegalovirus(CMV) immediate early promoter/enhancer, the Rous sarcoma virus longterminal repeat (RSV-LTR) or the SV-40 promoter.

Alternately, a tissue-specific promoter (a promoter that ispreferentially activated in a particular type of tissue and results inexpression of a gene product in that tissue) can be used in the vector.Such promoters include but are not limited to a liver specific promoter(Ill C R, et al., Blood Coagul Fibrinolysis 8 Suppl 2:S23-30, 1997) andthe EF-1 alpha promoter (Kim D W et al. Gene. 91(2):217-23, 1990, Guo ZS et al. Gene Ther. 3(9):802-10, 1996; U.S. Pat. Nos. 5,266,491 and5,225,348, each of which expressly incorporated by reference herein).Inducible promoters also find utility in practicing the methodsdescribed herein, such as a promoter containing the tet responsiveelement (TRE) in the tet-on or tet-off system as described (ClonTech andBASF), the metallothienein promoter which can be upregulated by additionof certain metal salts and rapamycin inducible promoters (Rivera et al.,1996, Nature Med, 2(9): 1028-1032; Ye et al., 2000, Science 283: 88-91;Sawyer T K et al., 2002, Mini Rev Med Chem. 2(5):475-88). Large numbersof suitable tissue-specific or regulatable vectors and promoters for usein practicing the current invention are known to those of skill in theart and many are commercially available.

Exemplary vector systems for use in practicing the invention include theretroviral MFG vector, described in U.S. Pat. No. 5,637,483, expresslyincorporated by reference herein. Other useful retroviral vectorsinclude pLJ, pEm and [alpha]SGC, described in U.S. Pat. No. 5,637,483(in particular Example 12), U.S. Pat. Nos. 6,506,604, 5,955,331 and U.S.Ser. No. 09/612,808, each of which is expressly incorporated byreference herein.

Further exemplary vector systems for use in practicing the inventioninclude second, third and fourth generation lentiviral vectors, U.S.Pat. Nos. 6,428,953, 5,665,577 and 5,981,276 and WO 00/72686, each ofwhich is expressly incorporated by reference herein.

Additional exemplary vector systems for use in practicing the presentinvention include adenoviral vectors, described for example in U.S. Pat.No. 5,872,005 and International Patent Publication No. WO 00/72686, eachof which is expressly incorporated by reference herein.

Yet another vector system that is preferred in practicing the methodsdescribed herein is a recombinant adeno-associated vector (rAAV) system,described for example in International Patent Publication Nos. WO98/46728 and WO 00/72686, Samulski et al., Virol. 63:3822-3828 (1989)and U.S. Pat. Nos. 5,436,146, 5,753,500, 6,037,177, 6,040,183 and6,093,570, each of which is expressly incorporated by reference herein.

Cytokines

Cytokines and combinations of cytokines have been shown to play animportant role in the stimulation of the immune system. The term“cytokine” is understood by those of skill in the art, as referring toany immunopotentiating protein (including a modified protein such as aglycoprotein) that enhances or modifies the immune response to a tumorpresent in the host. The cytokine typically enhances or modifies theimmune response by activating or enhancing the activity of cells of theimmune system and is not itself immunogenic to the host.

It follows from the results presented herein that a variety of cytokineswill find use in the present invention. Exemplary cytokines for use inpracticing the invention include but are not limited to interferon-alpha(IFN-α), interferon-beta (IFN-β), and interferon-gamma (IFN-γ),interleukins (e.g., IL-1 to IL-29, in particular, IL-2, IL-7, IL-12,IL-15 and IL-18), tumor necrosis factors (e.g., TNF-alpha and TNF-beta),erythropoietin (EPO), MIP3a, intracellular adhesion molecule (ICAM),macrophage colony stimulating factor (M-CSF), granulocyte colonystimulating factor (G-CSF) and granulocyte-macrophage colony stimulatingfactor (GM-CSF). The cytokine may be from any source, however, optimallythe cytokine is of murine or human origin (a native human or murinecytokine) or is a sequence variant of such a cytokine, so long as thecytokine has a sequence with substantial homology to the human form ofthe cytokine and exhibits a similar activity on the immune system. Itfollows that cytokines with substantial homology to the human forms ofIFN-alpha, IFN-beta, and IFN-gamma, IL-1 to IL-29, TNF-alpha, TNF-beta,EPO, MIP3a, ICAM, M-CSF, G-CSF and GM-CSF are useful in practicing theinvention, so long as the homologous form exhibits the same or a similareffect on the immune system. Proteins that are substantially similar toany particular cytokine, but have relatively minor changes in proteinsequence find use in the present invention. It is well known that smallalterations in protein sequence may not disturb the functional activityof a protein molecule, and thus proteins can be made that function ascytokines in the present invention but differ slightly from currentknown or native sequences.

Variant Sequences

Homologues and variants of native human or murine cytokines are includedwithin the scope of the invention. As used herein, the term “sequenceidentity” means nucleic acid or amino acid sequence identity between twoor more aligned sequences and is typically expressed as a percentage(“%”). The term “% homology” is used interchangeably herein with theterm “% identity” or “% sequence identity” and refers to the level ofnucleic acid or amino acid sequence identity between two or more alignedsequences, when aligned using a sequence alignment program. For example,as used herein, 80% homology has the same meaning as 80% sequenceidentity as determined by a defined algorithm, and accordingly ahomologue of a given sequence typically has greater than 80% sequenceidentity over a length of the given sequence. Preferred levels ofsequence identity include, but are not limited to, 80, 85, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98 or 99% or more sequence identity to anative cytokine amino acid or nucleic acid sequence, as describedherein.

Exemplary computer programs that can be used to determine the degree ofidentity between two sequences include, but are not limited to, thesuite of BLAST programs, e.g., BLASTN, BLASTX, TBLASTX, BLASTP andTBLASTN, all of which are publicly available on the Internet. See, also,Altschul, S. F. et al. Mol. Biol. 215:403-410, 1990 and Altschul, S. F.et al. Nucleic Acids Res. 25:3389-3402, 1997, expressly incorporated byreference herein. Sequence searches are typically carried out using theBLASTN program when evaluating a given nucleic acid sequence relative tonucleic acid sequences in the GenBank DNA Sequences and other publicdatabases. The BLASTX program is preferred for searching nucleic acidsequences that have been translated in all reading frames against aminoacid sequences in the GenBank Protein Sequences and other publicdatabases. In determining sequence identity, both BLASTN and BLASTX(i.e. version 2.2.5) are run using default parameters of an open gappenalty of 11.0, and an extended gap penalty of 1.0, and utilize theBLOSUM-62 matrix. [See, Altschul, et al., 1997, supra.] A preferredalignment of selected sequences in order to determine “% identity”between two or more sequences, is performed using for example, theCLUSTAL-W program in Mac Vector version 6.5, operated with defaultparameters, including an open gap penalty of 10.0, an extended gappenalty of 0.1, and a BLOSUM 30 similarity matrix.

A nucleic acid sequence is considered to be “selectively hybridizable”to a reference nucleic acid sequence if the two sequences specificallyhybridize to one another under moderate to high stringency hybridizationand wash conditions. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe. Forexample, “maximum stringency” typically occurs at about TM-5° C. (5°below the Tm of the probe) “high stringency” at about 5-10° below theTm; “intermediate stringency” at about 10-20° below the Tm of the probe;and “low stringency” at about 20-25° below the Tm. Functionally, maximumstringency conditions may be used to identify sequences having strictidentity or near-strict identity with the hybridization probe, whilehigh stringency conditions are used to identify sequences having about80% or more sequence identity with the probe. An example of highstringency conditions includes hybridization at about 42° C. in 50%formamide, 5×SSC, 5×Denhardt's solution, 0.5% SDS and 100 fig/mldenatured carrier DNA followed by washing two times in 2×SSC and 0.5%SDS at room temperature and two additional times in 0.1×SSC and 0.5% SDSat 42° C. Moderate and high stringency hybridization conditions are wellknown in the art. See, for example, Sambrook, et al, 1989, Chapters 9and 11, and in Ausubel, F. M., et al., 1993, (expressly incorporated byreference herein).

Anti-PD-1 Antibodies

As detailed herein, the present invention is directed to a method ofimproving an individual's immune response to cancer (e.g., a targetcancer antigen or antigens) by co-administering a cytokine-expressingcellular immunotherapy (e.g., GM-CSF) and at an antibody whichspecifically binds to human Programmed Death (PD)-1 to a patient withcancer.

PD-1 is an immunoinhibitory receptor belonging to the CD28 family(Freeman et al., J. Exp. Med. 192: 1027 (2000); Okazaki et al., Curr.Opim. Immunol. 14: 779 (2002)) and binds to two ligands, PD-L1 andPD-L2. PD-1 is induced on T-cells, B-cells and myeloid cells in-vitro(Agata et al., Int. Immunol. 8:765 (1996)), but is predominantlyexpressed on previously activated T-cells in vivo (Iwai et al., Immunol.Lett. 83:215 (2002)).

Studies indicate that PD-1 plays a critical role in immune responses.Engagement of PD-1 by PD-L1 leads to inhibition of T cell proliferationand cytokine production such as IL-2 and IFN-gamma (Freeman et al., J.Exp. Med. 192: 1027 (2000). In addition, PD-1 deficient mice exhibit abreakdown of peripheral tolerance and develop systemic autoimmunedisease (Nishimura et al., Immunity 11: 141-151 (1999); Nishimura etal., Science 291: 319-322 (2001)). Over-expression of PD-L1 has beenobserved in numerous human cancers, including melanomas and carcinomasof lung, ovary, colon, bladder, breast, cervix, liver, and head andneck, and glioblastoma (Dong et al., Nat. Med. 8:793-800 (2002); Brownet al., J. Immunol. 170:1257-66 (2003); Strome et al, Cancer Res. 63:6501 (2003); Wintterle et al., Cancer Res. 63:7462-7467 (2003)), andPD-L1/PD-1 interaction has been suggested to play a pivotal role in theimmune evasion of tumors from the host immune system (Blank et al.Cancer Immunol. Immunother. 54(4):307-14 (2005)). Therefore, blockade ofPD-L1/PD-1 interaction, e.g., with an antibody which specifically bindsPD-1, serves as one possible mechanism for enhancing anti-tumorimmunity.

Exemplary anti-PD-1 antibodies and methods for their use are describedby Goldberg et al., Blood 110(1):186-192 (2007), Thompson et al., Clin.Cancer Res. 13(6):1757-1761 (2007), and Korman et al., InternationalApplication No. PCT/JP2006/309606 (publication no. WO 2006/121168 A1),each of which are expressly incorporated by reference herein.

The antibodies for use in the present invention include, but are notlimited to, monoclonal antibodies, synthetic antibodies, polyclonalantibodies, multispecific antibodies (including bi-specific antibodies),human antibodies, humanized antibodies, chimeric antibodies,single-chain Fvs (scFv) (including bi-specific scFvs), single chainantibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fvs(sdFv), and epitope-binding fragments of any of the above. Inparticular, antibodies for use in the present invention includeimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain a PD-1 bindingsite that immunospecifically binds to PD-1. The immunoglobulin moleculesfor use in the invention can be of any type (e.g., IgG, IgE, IgM, IgD,IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) orsubclass of immunoglobulin molecule. Preferably, the antibodies for usein the invention are IgG, more preferably, IgG1.

The antibodies for use in the invention may be from any animal originincluding birds and mammals (e.g., human, murine, donkey, sheep, rabbit,goat, guinea pig, camel, horse, or chicken). Preferably, the antibodiesare human or humanized monoclonal antibodies. As used herein, “human”antibodies include antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or from mice or other animals that express antibodies fromhuman genes.

The antibodies for use in the present invention may be monospecific,bispecific, trispecific or of greater multispecificity. Multispecificantibodies may immunospecifically bind to different epitopes of apolypeptide or may immunospecifically bind to both a polypeptide as wella heterologous epitope, such as a heterologous polypeptide or solidsupport material. See, e.g., International Publication Nos. WO 93/17715,WO 92/08802, WO 91/00360, and WO 92/05793; Tutt, et al., 1991, J.Immunol. 147:60-69; U.S. Pat. Nos. 4,474,893, 4,714,681, 4,925,648,5,573,920, and 5,601,819; and Kostelny et al., 1992, J. Immunol.148:1547-1553.

The antibodies for use in the invention include derivatives of theantibodies. Standard techniques known to those of skill in the art canbe used to introduce mutations in the nucleotide sequence encoding anantibody to be used with the methods for use in the invention,including, for example, site-directed mutagenesis and PCR-mediatedmutagenesis which result in amino acid substitutions. Preferably, thederivatives include less than 25 amino acid substitutions, less than 20amino acid substitutions, less than 15 amino acid substitutions, lessthan 10 amino acid substitutions, less than 5 amino acid substitutions,less than 4 amino acid substitutions, less than 3 amino acidsubstitutions, or less than 2 amino acid substitutions relative to theoriginal molecule. In a preferred embodiment, the derivatives haveconservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a side chain with a similar charge. Familiesof amino acid residues having side chains with similar charges have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively,mutations can be introduced randomly along all or part of the codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for biological activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedand the activity of the protein can be determined.

The antibodies for use in the present invention include derivatives thatare modified, i.e., by the covalent attachment of any type of moleculeto the antibody. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, synthesis in the presence of tunicamycin, etc.Additionally, the derivative may contain one or more non-classical aminoacids.

The present invention also provides antibodies for use in the inventionthat comprise a framework region known to those of skill in the art. Incertain embodiments, one or more framework regions, preferably, all ofthe framework regions, of an antibody to be used in the compositions andmethods for use in the invention are human. In certain other embodimentsfor use in the invention, the fragment region of an antibody for use inthe invention is humanized. In certain embodiments, the antibody to beused with the methods for use in the invention is a synthetic antibody,a monoclonal antibody, an intrabody, a chimeric antibody, a humanantibody, a humanized chimeric antibody, a humanized antibody, aglycosylated antibody, a multispecific antibody, a human antibody, asingle-chain antibody, or a bispecific antibody.

In certain embodiments, an antibody for use in the invention has a highbinding affinity for PD-1. In specific embodiments, an antibody for usein the invention has an association rate constant or k_(on) rate ofabout 10⁵ M-1 s-1 or more, about 5×10⁵ M-1 s-1 or more, about 10⁶ M-1s-1 or more, about 5×10⁶ M-1 s-1 or more, about 10⁷ M-1 s-1 or more,about 5×10⁷ M-1 s-1 or more, about 10⁸ M-1 s-1 or more, about 5×10⁸ M-1s-1 or more, or about 1×10⁹ M-1 s-1 or more.

In other embodiments, an antibody for use in the invention has a k_(off)rate for PD-1 of about 5×10⁻¹ s-1 or less, about 10⁻¹ s-1 or less, about5×10⁻² s-1 or less, about 10⁻² s-1 or less, about 5×10⁻³ s-1 or less,about 10⁻³ s-1 or less, about 5×10⁻⁴ s-1 or less, about 10⁻⁴ s-1 orless, about 5×10⁻⁵ s-1 or less, about 10⁻⁵ s-1 or less, about 5×10⁻⁶ s-1or less, about 10⁻⁶ s-1 or less, about 5×10⁻⁷ s-1 or less, about 10⁻⁷s-1 or less, about 5×10⁻⁸ s-1 or less, about 10⁻⁸ s-1 or less, about5×10⁻⁹ s-1 or less, about 10⁻⁹ s-1 or less, about 5×10⁻¹⁰ s-1 or less,or about 10⁻¹⁰ s-1 or less.

In certain embodiments, an antibody for use in the invention has anaffinity constant or K_(a) (k_(on)/k_(off)) for PD-1 of about 10² M-1 ormore, about 5×10² M-1 or more, about 10³ M-1 or more, about 5×10³ M-1 ormore, about 10⁴ M-1 or more, about 5×10⁴ M-1 or more, about 10⁵ M-1 ormore, about 5×10⁵ M-1 or more, about 10⁶ M-1 or more, about 5×10⁶ M-1 ormore, about 10⁷ M-1 or more, about 5×10⁷ M-1 or more, about 10⁸ M-1 ormore, about 5×10⁸ M-1 or more, about 10⁹ M-1 or more, about 5×10⁹ M-1 ormore, about 10¹⁰ M-1 or more, about 5×10¹⁰ M-1 or more, about 10¹¹ M-1or more, about 5×10¹¹ M-1 or more, about 10¹² M-1 or more, about 5×10¹²M-1 or more, about 10¹³ M-1 or more, about 5×10¹³ M-1 or more, about10¹⁴ M-1 or more, about 5×10¹⁴ M-1 or more, about 10¹⁵ M-1 or more, orabout 5×10¹⁵ M-1 or more.

In certain embodiments, an antibody for use in the invention has a lowdissociation constant. In specific embodiments, the antibody-bindingdomain of a carrier construct for use in the invention has adissociation constant or K_(d) (k_(off)/k_(on)) for antibody about5×10⁻¹ M or less, about 10⁻¹ M or less, about 5×10⁻² M or less, about10⁻² M or less, about 5×10⁻³ M or less, about 10⁻³ M or less, about5×10⁻⁴ M or less, about 10⁻⁴ M or less, about 5×10⁻⁵ M or less, about10⁻⁵ M or less, about 5×10⁻⁶ M or less, about 10⁻⁶ M or less, about5×10⁻⁷ M or less, about 10⁻⁷ M or less, about 5×10⁻⁸ M or less, about10⁻⁸ M or less, about 5×10⁻⁹ M or less, about 10⁻⁹ M or less, about5×10⁻¹⁰ M or less, or about 10⁻¹⁰ M or less.

In certain embodiments, an antibody for use in the present invention hasa median effective concentration (EC₅₀) of less than 0.01 nM, less than0.025 nM, less than 0.05 nM, less than 0.1 nM, less than 0.25 nM, lessthan 0.5 nM, less than 0.75 nM, less than 1 nM, less than 1.25 nM, lessthan 1.5 nM, less than 1.75 nM, or less than 2 nM, in an in vitromicroneutralization assay. The median effective concentration is theconcentration of antibody that neutralizes 50% of PD-1 in an in vitromicroneutralization assay.

In certain embodiments, an antibody for use in the invention has ahalf-life in a subject, preferably a human, of about 12 hours or more,about 1 day or more, about 3 days or more, about 6 days or more, about10 days or more, about 15 days or more, about 20 days or more, about 25days or more, about 30 days or more, about 35 days or more, about 40days or more, about 45 days or more, about 2 months or more, about 3months or more, about 4 months or more, or about 5 months or more.Antibodies with increased in vivo half-lives can be generated bytechniques known to those of skill in the art. For example, antibodieswith increased in vivo half-lives can be generated by modifying (e.g.,substituting, deleting or adding) amino acid residues identified asinvolved in the interaction between the Fc domain and the FcRn receptor(see, e.g., International Publication No. WO 97/34631 and U.S. patentapplication Ser. No. 10/020,354, entitled “Molecules with ExtendedHalf-Lives, Compositions and Uses Thereof”, filed Dec. 12, 2001, byJohnson et al.; and U.S. Publication Nos. 2005/003700 and 2005/0064514,which are incorporated herein by reference in their entireties). Suchantibodies can be tested for binding activity to antigens as well as forin vivo efficacy using methods known to those skilled in the art, forexample, by immunoassays described herein.

Further, antibodies with increased in vivo half-lives can be generatedby attaching to the antibodies polymer molecules such as high molecularweight polyethyleneglycol (PEG). PEG can be attached to the antibodieswith or without a multifunctional linker either through site-specificconjugation of the PEG to the N- or C-terminus of the antibodies or viaepsilon-amino groups present on lysine residues. Linear or branchedpolymer derivatization that results in minimal loss of biologicalactivity will be used. The degree of conjugation will be closelymonitored by SDS-PAGE and mass spectrometry to ensure proper conjugationof PEG molecules to the antibodies. Unreacted PEG can be separated fromantibody-PEG conjugates by, e.g., size exclusion or ion-exchangechromatography. PEG-derivatized antibodies can be tested for bindingactivity to antigens as well as for in vivo efficacy using methods knownto those skilled in the art, for example, by immunoassays describedherein.

In certain embodiments, an antibody for use in the present inventionincludes antigen-binding portions of an intact antibody that retaincapacity to bind PD-1. Examples include (i) a Fab fragment, a monovalentfragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment consisting ofthe VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VHdomains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,(1989) Nature 341:544-546), which consists of a VH domain; and (vi) anisolated complementarity determining region (CDR). Furthermore, althoughthe two domains of the Fv fragment, VL and VH, are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair to form monovalent molecules (known as singlechain Fv (scFv); See, e.g., Bird et al. (1988) Science 242:423-426; andHuston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Suchsingle chain antibodies are included by reference to the term“antibody.”

In certain embodiments, the methods of the invention utilize combinationimmunotherapies that comprise additional molecules that have thecapacity to bind PD-1 and/or antagonize PD-1 function. A potential PD-1antagonist may be a protein closely related to a ligand of PD-1, forexample, a mutated form of PD-L1 or PD-L2, that recognizes the PD-1receptor but imparts no signaling effect, thereby competitivelyinhibiting the immunoinhibitory action of the ligand. Other potentialPD-1 antagonists include small molecules that bind to the receptorbinding site or other relevant binding site of PD-1, thereby blockingits normal biological activity. Examples of small molecules include, butare not limited to, small peptides or peptide-like molecules, preferablysoluble peptides, and synthetic non-peptidyl organic or inorganiccompounds. These small molecules can be identified by screeningtechniques well known for those skilled in the art.

Other antagonists may include oligonucleotides that bind toPD-1-encoding nucleic acids, such as antisense RNA or DNA constructsprepared using antisense technology, where, e.g., an antisense RNA orDNA molecule acts to block directly the translation of PD-1 mRNA byhybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes a maturetumor antigen herein, is used to design an antisense RNA oligonucleotideof from about 10 to 40 base pairs in length. A DNA oligonucleotide isdesigned to be complementary to a region of the gene involved intranscription (triple helix-see, Lee et al., Nucl. Acids Res., 6:3073(1979); Cooney et al., Science, 241: 456 (1988); Dervan et al., Science,251:1360 (1991)), thereby preventing transcription and the production ofthe target polypeptide. The antisense RNA oligonucleotide hybridizes tothe mRNA in vivo and blocks translation of the mRNA molecule into thetarget polypeptide (antisense-Okano, Neurochem., 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRCPress: Boca Raton, Fla., 1988).

The oligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of the PD-1 polypeptide. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation-initiation site,e.g., between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

Cytokine-Expressing Cellular Immunotherapy

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokineproduced by fibroblasts, endothelial cells, T cells and macrophages.This cytokine has been shown to induce the growth of hematopoetic cellsof granulocyte and macrophage lineages. In addition, the cytokineactivates the antigen processing and presenting function of dendriticcells, which are the major antigen presenting cells (APC) of the immunesystem. Results from animal model experiments have convincingly shownthat GM-CSF producing tumor cells are able to induce an immune responseagainst parental, non-transduced tumor cells.

Autologous and allogeneic cancer cells that have been geneticallymodified to express a cytokine, e.g., GM-CSF, followed byreadministration to a patient for the treatment of cancer are describedin U.S. Pat. Nos. 5,637,483, 5,904,920 and 6,350,445, expresslyincorporated by reference herein. A form of GM-CSF-expressinggenetically modified cancer cells or a “cytokine-expressing cellularimmunotherapy” for the treatment of pancreatic cancer is described inU.S. Pat. Nos. 6,033,674 and 5,985,290, expressly incorporated byreference herein. A universal immunomodulatory cytokine-expressingbystander cell line is described in U.S. Pat. No. 6,464,973, expresslyincorporated by reference herein. Clinical trials employingGM-CSF-expressing autologous or allogeneic cellular immunotherapies havebeen undertaken for treatment of prostate cancer, melanoma, lung cancer,pancreatic cancer, renal cancer, and multiple myeloma. A number of thesetrials are currently ongoing, however, and the question still remainsopen as to whether the immune response to GM-CSF expressing cells alonewill be sufficiently powerful to slow or eradicate large or fast growingmalignancies.

The present invention provides an improved method of stimulating animmune response to cancer in a mammalian, preferably a human, subject.Desirably, the method effects a systemic immune response, i.e., a T-cellresponse and/or a B-cell response, to the cancer. In some embodiments,the method comprises administering to the patient a cytokine-expressingcellular immunotherapy and an antibody that specifically binds to PD-1,wherein the cellular immunotherapy comprises cells which express acancer antigen or various cancer antigens. The cancer antigen/antigenscan be one of the antigens of the cancer found in the patient undertreatment. The cells can be rendered proliferation incompetent, such ase.g., by irradiation. Upon administration of the cytokine-expressingcellular immunotherapy and the anti-PD-1 antibody, an immune response tothe cancer can be elicited or enhanced.

In one approach, the cytokine expressing cellular immunotherapycomprises a single population of cells that is modified to express acytokine, e.g. GM-CSF. In another approach, the immunotherapy comprisesa single population of cells that is modified to express a cytokine aswell as a single chain antibody that specifically binds to PD-1. Inanother approach, the immunotherapy comprises a combination of two ormore populations of cells individually modified to express one componentof the immunotherapy, e.g. a cytokine and a single chain anti-PD-1antibody.

In general, a cytokine-expressing cellular immunotherapy for use inpracticing the invention comprises tumor cells selected from the groupconsisting of autologous tumor cells, allogeneic tumor cells and tumorcell lines (i.e., bystander cells). In one aspect of the invention, thecells of the cytokine-expressing cellular immunotherapy are administeredto the same individual from whom they were derived (autologous). Inanother aspect of the invention, the cells of the cytokine-expressingcellular immunotherapy and the tumor are derived from differentindividuals (allogeneic or bystander). By way of example, in oneapproach, genetically modified GM-CSF expressing tumor cells areprovided as an allogeneic or bystander cell line and one or moreadditional cancer therapeutic agents, e.g. an anti-PD-1 antibody, isincluded in the treatment regimen. In another approach, one or moreadditional transgenes are expressed by an allogeneic or bystander cellline while a cytokine (i.e., GM-CSF) is expressed by autologous orallogeneic cells, and one or more additional cancer therapeutic agents,e.g. an anti-PD-1 antibody, is included in the treatment regimen.

In a preferred approach, the tumor being treated is selected from thegroup consisting of cancer of the bladder, breast, colon, kidney, liver,lung, ovary, cervix, pancreas, rectum, prostate, stomach, epidermis, ahematopoietic tumor of lymphoid or myeloid lineage, a tumor ofmesenchymal origin such as a fibrosarcoma or rhabdomyosarcoma, melanoma,teratocarcinoma, neuroblastoma, glioma, adenocarcinoma and non-smalllung cell carcinoma.

In previous studies, a direct comparison of murine tumor cellstransduced with various cytokines demonstrated that GM-CSF-secretingtumor cells induced the best overall anti-tumor protection. In onepreferred embodiment, the cytokine expressed by the cytokine-expressingcellular immunotherapy of the invention is GM-CSF. The preferred codingsequence for GM-CSF is the genomic sequence described in Huebner K. etal, Science 230(4731): 1282-5, 1985. Alternatively the cDNA form ofGM-CSF finds utility in practicing the invention (Cantrell et al., Proc.Natl. Acad. Sci., 82, 6250-6254, 1985).

In some embodiments, the cells of the cytokine-expressing cellularimmunotherapy are cryopreserved prior to administration. Prior toadministration, the cells of a cytokine-expressing cellularimmunotherapy of the invention are rendered proliferation incompetent.While a number of means of rendering cells proliferation incompetent areknown, irradiation is the preferred method. Preferably, thecytokine-expressing cellular immunotherapy combination is irradiated ata dose of from about 50 to about 200 rads/min, even more preferably,from about 120 to about 140 rads/min prior to administration to thepatient. Most importantly, the cells are irradiated with a totalradiation dose sufficient to inhibit growth of substantially 100% of thecells, from further proliferation. Thus, desirably the cells areirradiated with a total dose of from about 10,000 to 20,000 rads,optimally, with about 15,000 rads.

Typically more than one administration of cytokine (e.g., GM-CSF)producing cells is delivered to the subject in a course of treatment.Dependent upon the particular course of treatment, multiple injectionsmay be given at a single time point with the treatment repeated atvarious time intervals. For example, an initial or “priming” treatmentmay be followed by one or more “booster” treatments. Such “priming” and“booster” treatments are typically delivered by the same route ofadministration and/or at about the same site. When multiple doses areadministered, the first immunization dose may be higher than subsequentimmunization doses. For example, a 5×10⁶ prime dose may be followed byseveral booster doses of 10⁶ to 3×10⁶ GM-CSF producing cells.

A single injection of cytokine-producing cells is typically betweenabout 10⁶ to 10⁸ cells, e.g., 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶,7×10⁶, 8×10⁶, 9×10⁶, 10⁷, 2×10⁷, 5×10⁷, or as many as 10⁸ cells. In oneembodiment, there are between 10⁶ and 10⁸ cytokine-producing cells perunit dose. The number of cytokine-producing cells may be adjustedaccording, for example, to the level of cytokine produced by a givencytokine producing cellular immunotherapy.

Autologous

The use of autologous cytokine-expressing cells in a immunotherapy ofthe invention provides advantages since each patient's tumor expresses aunique set of tumor antigens that can differ from those found onhistologicaily-similar, MHC-matched tumor cells from another patient.See, e.g., Kawakami et al, J. Immunol, 148, 638-643 (1992); Darrow etal., J. Immunol., 142, 3329-3335 (1989); and Horn et al., J.Immunother., 10, 153-164 (1991). In contrast, MHC-matched tumor cellsprovide the advantage that the patient need not be taken to surgery toobtain a sample of their tumor for immunotherapy production.

In one preferred aspect, the present invention comprises a method oftreating cancer by carrying out the steps of: (a) obtaining tumor cellsfrom a mammal, preferably a human, harboring a tumor; (b) modifying thetumor cells to render them capable of producing a cytokine or anincreased level of a cytokine naturally produced by the cells relativeto unmodified tumor cells; (c) rendering the modified tumor cellsproliferation incompetent; and (d) readministering the modified tumorcells to the mammal from which the tumor cells were obtained or to amammal with the same MHC type as the mammal from which the tumor cellswere obtained, in combination with administration of an anti-PD-1antibody. The administered tumor cells are autologous or MHC-matched tothe host.

Allogeneic

Researchers have sought alternatives to autologous and MHC-matched cellsas tumor immunotherapies, as reviewed by Jaffee et al., Seminars inOncology, 22, 81-91 (1995). Early tumor immunotherapy strategies werebased on the understanding that the vaccinating tumor cells function asthe antigen presenting cells (APCs) and present tumor antigens by way oftheir MHC class I and II molecules, and directly activate the T cell armof the immune system. The results of Huang et al. (Science, 264,961-965, 1994), indicate that professional APCs of the host rather thanthe vaccinating tumor cells prime the T cell arm of the immune system bysecreting cytokine(s) such as GM-CSF such that bone marrow-derived APCsare recruited to the region of the tumor. These results suggest that itmay not be necessary or optimal to use autologous or MHC-matched tumorcells in order to elicit an anti-cancer immune response and that thetransfer of allogeneic MHC genes (from a genetically dissimilarindividual of the same species) can enhance tumor immunogenicity. Morespecifically, in certain cases, the rejection of tumors expressingallogeneic MHC class I molecules resulted in enhanced systemic immuneresponses against subsequent challenge with the unmodified parentaltumor, as reviewed in Jaffee et al., supra, and Huang et al., supra.

In one preferred aspect, the invention provides a method for treatingcancer by carrying out the steps of: (a) obtaining a tumor cell line;(b) modifying the tumor cell line to render the cells capable ofproducing an increased level of a cytokine relative to the unmodifiedtumor cell line; (c) rendering the modified tumor cell lineproliferation incompetent; and (d) administering the tumor cell line toa mammalian host having at least one tumor that is the same type oftumor as that from which the tumor cell line was obtained or wherein thetumor cell line and host tumor express at least one common antigen, incombination with administration of an anti-PD-1 antibody. Theadministered tumor cell line is allogeneic and is not MHC-matched to thehost. Such allogeneic lines provide the advantage that they can beprepared in advance, characterized, aliquoted in vials containing knownnumbers of cytokine-expressing cells and stored such thatwell-characterized cells are available for administration to thepatient. Methods for the production of gene-modified allogeneic cellsare described for example in International Patent Publication No. WO00/72686A1, expressly incorporated by reference herein.

In one approach to preparing a cytokine-expressing cellularimmunotherapy comprising gene-modified allogeneic cells,cytokine-encoding nucleic acid sequences are introduced into a cell linethat is an allogeneic tumor cell line (i.e., derived from an individualother than the individual being treated). The tumor cell line may be ofthe same type as the tumor or cancer being treated. The tumor and/ortumor cell line may be from any form of cancer, including, but notlimited to, carcinoma of the bladder, breast, colon, kidney, liver,lung, ovary, cervix, pancreas, rectum, prostate, stomach, epidermis, ahematopoietic tumor of lymphoid or myeloid lineage, a tumor ofmesenchymal origin such as a fibrosarcoma or rhabdomyosarcoma, melanoma,teratocarcinoma, neuroblastoma, glioma, adenocarcinoma, and non-smalllung cell carcinoma.

Desirably, the allogeneic cell line expresses GM-CSF in a range from200-1000 ng/10⁶ cells/24 h. Preferably, the universal bystander cellline expresses at least about 200 ng GM-CSF/10⁶ cells/24 hours.

In practicing the invention, one or more allogeneic cell lines can beincubated with an autologous cancer antigen, e.g., an autologous tumorcell (which together comprise an allogeneic cell line composition), thenthe allogeneic cell line composition can be administered to the patient.Typically, the cancer antigen can be provided by (on) a cell of thecancer to be treated, i.e., an autologous cancer cell. In such cases,the composition can be rendered proliferation-incompetent byirradiation, wherein the allogeneic cells and cancer cells are plated ina tissue culture plate and irradiated at room temperature using a Cssource, as detailed above. The ratio of allogeneic cells to autologouscancer cells in a given administration will vary dependent upon thecombination.

Any suitable route of administration can be used to introduce anallogeneic cell line composition into the patient. Preferably, thecomposition is administered subcutaneously or intratumorally.

The use of allogeneic cell lines in practicing the present inventionenables administration of a cytokine-expressing allogeneic cell line toa patient with cancer, together with an autologous cancer antigen. Thistreatment can result in an effective immune response to a tumor. Thisapproach advantageously obviates the need to culture and transduceautologous tumor cells for each patient, eliminating the problem ofvariable and inefficient transduction efficiencies.

Bystander

In one further aspect, the present invention provides a universalimmunomodulatory cytokine-expressing bystander cell line and ananti-PD-1 antibody. The universal bystander cell line comprises cellswhich either naturally lack major histocompatibility class I (MHC-I)antigens and major histocompatibility class II (MHC-II) antigens or havebeen modified so that they lack MHC-I antigens and MHC-II antigens. Inone aspect of the invention, a universal bystander cell line is modifiedby introduction of a vector comprising a nucleic acid sequence encodinga cytokine operably linked to a promoter and expression controlsequences necessary for expression thereof. The nucleic acid sequenceencoding the cytokine may or may not further comprise a selectablemarker sequence operably linked to a promoter. The universal bystandercell line preferably grows in defined, e.g., serum-free, medium,preferably as a suspension.

An example of a preferred universal bystander cell line is K562 (ATCCCCL-243; Lozzio et al, Blood 45(3): 321-334 (1975); Klein et al., Int.J. Cancer 18: 421-431 (1976)). A detailed description of human bystandercell lines is described for example in U.S. Pat. No. 6,464,973 andInternational Patent Publication No. WO 9938954. Desirably, theuniversal bystander cell line expresses the cytokine, e.g., GM-CSF inthe range from 200-1000 ng/10⁶ cells/24 h. Preferably, the universalbystander cell line expresses at least about 200 ng GM-CSF/10⁶ cells/24hours.

In practicing the invention, the universal bystander cell lines can beincubated with an autologous cancer antigen, e.g., an autologous tumorcell (which together comprise a universal bystander cell linecomposition), then the universal bystander cell line composition can beadministered to the patient. Any suitable route of administration can beused to introduce a universal bystander cell line composition into thepatient. Preferably, the composition is administered subcutaneously orintratumorally.

Typically, the cancer antigen can be provided by (on) a cell of thecancer to be treated, i.e., an autologous cancer cell. In such cases,the composition can be rendered proliferation-incompetent byirradiation, wherein the bystander cells and cancer cells are plated ina tissue culture plate and irradiated at room temperature using a Cssource, as detailed above.

The ratio of bystander cells to autologous cancer cells in a givenadministration will vary dependent upon the combination. With respect toGM-CSF-producing bystander cells, the ratio of bystander cells toautologous cancer cells in a given administration should be such that atleast 36 ng GM-CSF/10⁶ cells/24 hrs is produced, as the therapeuticeffect may be decreased if the concentration of GM-CSF is less thanthis. In addition to the GM-CSF threshold, the ratio of bystander cellsto autologous cancer cells should not be greater than 1:1. Appropriateratios of bystander cells to tumor cells or tumor antigens can bedetermined using routine methods in the art.

The use of bystander cell lines in practicing the present inventionenables administration of a cytokine-expressing bystander cell line to apatient with cancer, together with an autologous cancer antigen. Thistreatment can result in an effective immune response to a tumor. Thisapproach advantageously obviates the need to culture and transduceautologous tumor cells for each patient, eliminating the problem ofvariable and inefficient transduction efficiencies.

Evaluation of Combination Immunotherapy in Animal Models B16F10 MelanomaModel

In one approach, the efficacy of a cytokine-expressing cellularimmunotherapy co-administered with an anti-PD-1 antibody can beevaluated by carrying out animal studies in the syngeneic B16F10melanoma tumor model in the treatment setting. See, e.g., Griswold D PJr., Cancer Chemother Rep 2; 3(1):315-24, 1972 and Berkelhammer J et al,Cancer Res 42(8):3157-63, 1982. The murine melanoma cell line B16 is awell-defined cell line which is weakly immunogenic in syngeneic C57B16mice and therefore readily forms tumors in C57BL6 mice. Furthermore,several tumor associated antigens have been identified in this modelwhich allow one to monitor tumor specific as well as antigen specificimmune responses. In addition, several murine-specific reagents arecommercially available and are used to monitor anti-tumor immuneresponses in the various immunotherapy strategies. A typical study inthe B16F10 melanoma tumor model makes use of at least 6 and generally10-15 mice per group in order to obtain statistically significantresults. Statistical significance can be evaluated using the Student'st-test.

Vaccination of C57BL/6 mice with irradiated GM-CSF-secreting B16F10tumor cells stimulates potent, long-lasting and specific anti-tumorimmunity that prevents tumor growth in most mice subsequently challengedwith wild-type B16F10 cells. However, this protection is less effectivewhen GM-CSF-producing tumor cell immunotherapies are administered tomice with preexisting tumor burden. In carrying out studies using theB16F10 melanoma tumor model, female C57BL/6 mice are obtained fromTaconic and are 6-8 weeks old at the start of each experiment. In atypical experiment, mice are injected with 1-2×10⁵ B16BF10 cells on day0 subcutaneously in a dorsal/anterior location. On day 3, mice arevaccinated in a ventral/posterior location with 1-3×10⁶ irradiated(10,000 rads) B16F10 or cytokine-expressing cellular immunotherapy. Miceare followed for tumor development and survival. After 14-21 days, miceare sacrificed and their tumor burden assessed by harvesting the micelungs and counting the surface tumor metastasis and measuring the weightof the lung. An alternative B16F10 melanoma tumor model involvessubcutaneous injection of B16F10 tumor cells. A typical in vivo study inthe B16F10 melanoma tumor model employs the following groups: HBSS only(negative control); cytokine-expressing cellular immunotherapy/HBSS;(cellular monotherapy control); anti-PD-1 antibody (antibody onlycontrol); cytokine-expressing cellular immunotherapy plus anti-PD-1antibody.

Previous experiments have demonstrated that HBSS or irradiated B16F10alone do not protect challenged mice from tumor formation.GM-CSF-expressing cellular immunotherapies alone were shown to protectfrom 10-20% of the challenged mice. The combination of acytokine-expressing cellular immunotherapy plus an anti-PD-1 antibody isexpected to increase the efficacy of anti-tumor protection. The degreeof protection depends on several factors, such as the expression levelof the cytokine-expressing cellular immunotherapy, the dosage and dosingfrequency of the anti-PD-1 antibody, and the relative timing ofadministration of the anti-PD-1 antibody relative to the timing ofadministration of the cytokine-expressing cellular immunotherapy.

Immunological Monitoring

Several tumor associated antigens have been identified which allow oneto monitor tumor specific as well as antigen-specific immune responses.For example, tumor antigen-specific T cells can be identified by therelease of IFN-gamma following antigenic restimulation in vitro (Hu,H-M. et al, Cancer Research, 2002, 62; 3914-3919) (FIGS. 2C, 3B). Yetanother example of new methods used to identify tumor antigen-specific Tcells is the development of soluble MHC I molecules also known as MHCtetramers (Beckman Coulter, Immunomics), reported to be loaded withspecific peptides shown to be involved in an anti-tumor immune response.(FIG. 2A).

Tetramer staining may be used to monitor tumor-specific T-cell responsesand to identify very low frequencies of antigen-specific T-cells.Because tetramer staining is performed on freshly isolated lymphocyteswithin several hours of removal, and without further in vitrostimulation, the technique can be used to estimate the frequency oftumor antigen-specific T-cells in vivo. This provides a means to comparethe potency of different tumor immunotherapy strategies.

Examples within the B16F10 melanoma tumor model include but are notlimited to gp100, Trp2, Trp-1, and tyrosinase. Similarmelanoma-associated antigens have been identified in humans. Such toolsprovide information that can then be translated into the clinical arena.

Assays for Efficacy of Combination Immunotherapy in In Vivo Models

Tumor burden can be assessed at various time points after tumorchallenge using techniques well known in the art. Assays for monitoringanti-tumor response and determining the efficacy of combinationimmunotherapy are described below. While an improved or enhancedanti-tumor immune response may be most dramatically observed shortlyfollowing administration of the immunotherapy, e.g. within 5-10 days,the response may be delayed in some instances, depending on factors suchas the expression level of the cytokine-expressing cellularimmunotherapy, the dosage and dosing frequency of the anti-PD-1antibody, and the relative timing of administration of the anti-PD-1antibody relative to the timing of administration of thecytokine-expressing cellular immunotherapy. Thus, the following assaysmay be performed on biological samples harvested at much later timepoints than is indicated below in order to fully assess the anti-tumorresponse following immunotherapy.

Cytotoxic T lymphocyte (CTL) activity may be determined both in vitroand in vivo. Typically, spleen cells are assessed for CTL activity by invitro whole cell stimulation for 5 days. Target cells are labeled with⁵¹Cr and co-incubated with splenic effector CTL, and release of ⁵¹Crinto the supernatants is an indicator of CTL lysis of target cells. Onday 3, in vitro stimulated CTL supernatants are tested for IFN-gammaproduction by CTL. In brief, wells are coated with coating antibodyspecific for IFN-gamma, supernatant is then added to wells, andIFN-gamma is detected using an IFN-gamma specific detecting antibody.IFN-gamma can also be detected by flow cytometry, in order to measurecell-specific IFN-gamma production.

In vivo CTL activity may be assessed via carboxyfluoroscein diacetatesuccinimidyl ester (CSFE) labeling of syngeneic splenocytes. In brief,splenocyte target populations are evenly split into two populations. Thefirst population is pulsed with antigen specific peptide, e.g. SIINFEKLpeptide from OVA, and labeled with a high concentration of CSFE, e.g.2.5 μM (CSFE^(hi)), while the second population is non-pulsed andlabeled with a low concentration of CSFE, e.g. 0.25 μM. (CSFE^(lo)). Anequal number of cells from each population are mixed together andinjected into mice immunized with cytokine expressing tumor cellimmunotherapy alone, or in combination with anti-PD-1 antibody. 18 hoursfollowing injection, splenocytes are harvested and cell suspensions areanalyzed by flow cytometry, and cell populations are distinguished basedon varying fluorescent intensities. Percent specific lysis is determinedby the loss of the peptide-pulsed CSFE^(hi) population compared to thecontrol CSFE^(lo) population. (FIG. 2B).

Another indication of an effective anti-tumor immune response is theproduction of effector cytokines such as TNF-alpha, IFN-gamma, IL-5,IL-6, IL-10 and monocyte chemotactic protein (MCP)-1 upon restimulationin vitro. Cytokine levels were measured in supernatants from spleencells restimulated in vitro for 48 hours with irradiatedGM-CSF-expressing cells. (FIG. 3)

A further method used to monitor tumor-specific T cell responses is viaintracellular cytokine staining (ICS). ICS can be used to monitortumor-specific T-cell responses and to identify very low frequencies ofantigen-specific T-cells. Because ICS is performed on freshly isolatedlymphocytes within 5 hours of removal, unlike the CTL and cytokinerelease assays, which often require 2-7 days of in vitro stimulation, itcan be used to estimate the frequency of tumor antigen-specific T-cellsin vivo. This provides a powerful technique to compare the potency ofdifferent tumor immunotherapy strategies. ICS has been used to monitorT-cell responses to melanoma-associated antigens such as gp 100 and Trp2following various melanoma immunotherapy strategies. Such T-cells can beidentified by the induction of intracellular IFN-gamma expressionfollowing stimulation with a tumor-specific peptide bound to MHC I.

Xenogen Imaging of Tumor Models

In some studies, in vivo luminescence of tumor bearing mice is monitoredby monitoring of B16F10-luciferase (Xenogen Inc.) injected mice. Inbrief, Balb/c nu/nu mice are injected with 5×10⁴ or 2×10⁵ cells ofB16F10-luc cells via tail vein on day 0. Mice are monitored for tumorburden when necessary by intra-peritoneal injection of excess luciferinsubstrate at 1.5 mg/g mice weight. In a typical analysis, twenty minutesafter substrate injection, mice are anesthesized and monitored for invivo luminescence with Xenogen IVIS Imaging System (Xenogen Inc.)luminescence sensitive CCD camera by dorsal or ventral position. Data iscollected and analyzed by Living Image 2.11 software.

Cytokine-Expressing Cellular Immunotherapies Plus Anti-PD-1 Antibodies

Previous reports indicate that GM-CSF-secreting tumor cell immunotherapyprovides partial protection of mice when used as a monotherapy fornon-immunogenic tumors such as B16 melanoma. The results presentedherein demonstrate that the combination of GM-CSF-secreting B16 tumorcells and an anti-PD-1 antibody can act synergistically, resulting inhighly protective antitumor immune responses. In order to achieve themaximal synergistic effect of these two agents in clinical trials,possible treatment regimens should be carefully evaluated in preclinicalstudies. In ongoing clinical trials GM-CSF-secreting tumor cellimmunotherapies or anti-PD-1 antibodies are administered to patientsrepeatedly over a period of several months. In studies described herein,the efficacy of the combination was evaluated in preclinical studiesfollowing repeated administration of both GM-CSF-secreting tumor cellimmunotherapies and anti-PD-1 antibodies. Example 4 details studieswhere a cytokine expressing cellular immunotherapy was tested in both aB16F10 and CT26 tumor model with and without co-administration of ananti-PD-1 antibody. The GM-CSF-secreting tumor cell immunotherapy wasless effective than the combination therapy of anti-PD-1 antibody andGM-CSF-secreting tumor cell immunotherapies (FIGS. 4, 5).

These results demonstrate that in practicing the present invention, anautologous, allogeneic, or bystander cytokine-expressing cellularimmunotherapy may be administered to a cancer patient in combinationwith an anti-PD-1 antibody, resulting in enhanced therapeutic efficacyand prolonged survival relative to either monotherapy alone.

In a preferred aspect of the methods described herein, acytokine-expressing cellular immunotherapy is administered incombination with an anti-PD-1 antibody to a cancer patient, wherein thecytokine-expressing cellular immunotherapy comprises mammalian,preferably human tumor cells, and the cells in the cytokine-expressingcellular immunotherapy are rendered proliferation incompetent, such asby irradiation. Administration of a cytokine-expressing cellularimmunotherapy in combination with an anti-PD-1 antibody results in anenhanced immune response to the cancer as compared to the immuneresponse to the same cancer following administration of thecytokine-expressing cellular immunotherapy or anti-PD-1 antibody alone.

The cytokine-expressing cellular immunotherapy combination may beadministered by any suitable route. Preferably, the composition isadministered subcutaneously or intratumorally. Local or systemicdelivery can be accomplished by administration comprising administrationof the combination into body cavities, by parenteral introduction,comprising intramuscular, intravenous, intraportal, intrahepatic,peritoneal, subcutaneous, or intradermal administration. In the eventthat the tumor is in the central nervous system, the composition isadministered in the periphery to prime naive T-cells in the draininglymph nodes. The activated tumor-specific T-cells are able to cross theblood/brain barrier to find their targets within the central nervoussystem.

In some embodiments of the combination immunotherapy described herein,the cells of the cytokine-expressing cellular immunotherapy and theanti-PD-1 antibody can be administered at essentially the same time,i.e., concurrently, e.g., within the same hour or same day, etc., or atseparately staggered times, i.e. sequentially prior to or subsequent tothe administration of the second agent of the combination immunotherapy,e.g., on separate days, weeks, etc. The instant methods are therefore tobe understood to include all such regimes of simultaneous ornon-simultaneous treatment. In some embodiments, the cells of thecytokine-expressing cellular immunotherapy are administered within 0.1,0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 ormore than 18 hours of administration of the anti-PD-1 antibody. In someembodiments, the cells of the cytokine-expressing cellular immunotherapyare administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 ormore than 14 days of administration of the anti-PD-1 antibody. In someembodiments, the cells of the cytokine-expressing cellular immunotherapyare administered within 1, 2, 3, 4, 5 or more than 5 weeks ofadministration of the anti-PD-1 antibody.

In some embodiments of the combination immunotherapy described herein,the cells of the cytokine-expressing cellular immunotherapy areadministered simultaneously with administration of the anti-PD-1antibody, for example, with the first administration of the combinationtherapy and with subsequent administrations of the combination therapy.In a particular embodiment, the cells of the cytokine-expressingcellular immunotherapy are administered simultaneously with the firstadministration of the anti-PD-1 antibody, and the cells of thecytokine-expressing cellular immunotherapy are administeredsimultaneously with administration of the anti-PD-1 antibody on abiweekly basis thereafter. In another particular embodiment, the cellsof the cytokine-expressing cellular immunotherapy are administeredwithin 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23 or 24 hours of administration of theanti-PD-1 antibody, and the cells of the cytokine-expressing cellularimmunotherapy are administered within 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours ofadministration of the anti-PD-1 antibody, on a biweekly basisthereafter.

In certain embodiments, cells of the cytokine-expressing cellularimmunotherapy are administered prior to administration of the anti-PD-1antibody. In some embodiments, the cells of the cytokine-expressingcellular immunotherapy are administered one, two, three, four, five,six, seven, or more days prior to administration of the anti-PD-1antibody. In other embodiments, cells of the cytokine-expressingcellular immunotherapy are administered after administration of theanti-PD-1 antibody. In some embodiments, the cells of thecytokine-expressing cellular immunotherapy are administered one, two,three, four, five, six, seven, or more days after administration of theanti-PD-1 antibody.

As will be understood by those of skill in the art, the optimaltreatment regimen will vary. As a result, it will be understood that thestatus of the cancer patient and the general health of the patient priorto, during, and following administration of a cytokine-expressingcellular immunotherapy in combination with an anti-PD-1 antibody, thepatient will be evaluated in order to determine if the dose of eachcomponent and relative timing of administration should be optimized toenhance efficacy or additional cycles of administration are indicated.Such evaluation is typically carried out using tests employed by thoseof skill in the art to evaluate traditional cancer chemotherapy, asfurther described below in the section entitled “Monitoring Treatment.”

Monitoring Treatment

One skilled in the art is aware of means to monitor the therapeuticoutcome and/or the systemic immune response upon administering acombination treatment of the present invention. In particular, thetherapeutic outcome can be assessed by monitoring attenuation of tumorgrowth and/or tumor regression and or the level of tumor specificmarkers. The attenuation of tumor growth or tumor regression in responseto treatment can be monitored using one or more of several end-pointsknown to those skilled in the art including, for instance, number oftumors, tumor mass or size, or reduction/prevention of metastasis.

Kits

The combination immunotherapy composition can be included in a kit,container, pack, or dispenser together with instructions foradministration. When the composition is supplied as a kit, the differentcomponents of the composition may be packaged in separate containers soas to permit long-term storage without losing the active components'functions. The reagents included in the kits can be supplied incontainers of any sort such that the life of the different componentsare preserved, and are not adsorbed or altered by the materials of thecontainer. For example, cytokine-expressing cells may be housed incontainers such as test tubes, vials, flasks, bottles, syringes, or thelike. Sealed glass ampules may be used to contain lyophilized anti-PD-1antibody that has been packaged under a neutral, non-reacting gas, suchas nitrogen. Ampoules may consist of any suitable material, such asglass, organic polymers, such as polycarbonate, polystyrene, etc.,ceramic, metal or any other material typically employed to holdreagents. Other examples of suitable containers include simple bottlesthat may be fabricated from similar substances as ampules, andenvelopes, that may consist of foil-lined interiors, such as aluminum oran alloy. Containers may have a sterile access port, such as a bottlehaving a stopper that can be pierced by a hypodermic injection needle.Other containers may have two compartments that are separated by areadily removable membrane that upon removal permits the components tomix. Removable membranes may be glass, plastic, rubber, etc.

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrate, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zipdisc, videotape, audio tape, etc. Detailed instructions may not bephysically associated with the kit; instead, a user may be directed toan internet web site specified by the manufacturer or distributor of thekit, or supplied as electronic mail.

Example 1 Immune Response Following Vaccination with a GM-CSF-SecretingImmunotherapy Combined with Administration of Anti-PD-1 Antibody

Immune responses were measured as increases in tumor antigen-specificT-cells. Such T-cells can be identified by immunological monitoringmethods, as described above, including (A) tetramer staining, (B) invivo CTL activity, and (C) the induction of intracellular IFN-gammaexpression following stimulation with a tumor-specific peptide or tumorcells.

FIG. 1A illustrates the anti-tumor T-cell response induced byadministration of GM-CSF-secreting B16F10 cells alone and in combinationwith an anti-PD-1 antibody, as determined by tetramer staining. On day0, 1×10⁶ OT-1 transgenic T-cells were adoptively transferred intotumor-bearing C57BL/6 mice which were challenged on day −1 with 2×10⁵live B16 cells transduced with the surrogate antigen, ovalbumin(F10.ova). On day 3, mice were immunized with 1×10⁶ irradiatedGM-CSF-secreting B16 cells expressing ovalbumin as a surrogate antigen(GM.ova) as immunotherapy alone, or immunotherapy was followed by 200 μgand 100 μg of anti-PD-1 antibody on days 3 and 4, respectively, ascombination therapy. At the indicated timepoints, draining lymph nodes(top panel) and spleens (bottom panel) were harvested from selected mice(n=5/group) and evaluated for antigen-specific T-cell count by tetramerstaining. Immunization with GM.ova plus anti-PD-1 antibody resulted inan increase in the number of antigen specific T-cells in both draininglymph nodes (DLN) and spleen compared to immunization with GM.ovaimmunotherapy alone. This increase was observed starting at 7 days postimmunotherapy and was sustained out to 20 days.

FIG. 1B illustrates the in vivo cytolytic activity of T-cells in micetreated with GM-CSF-secreting B16F10 cells alone and in combination withan anti-PD-1 antibody. On day 0, mice were inoculated with 2×10⁵ liveB16F10 cells transduced with the surrogate antigen, ovalbumin (F10.ova).On day 3, mice were immunized with 1×10⁶ irradiated GM-CSF-secretingF10.ova cells (GM.ova) as immunotherapy alone, or immunotherapy wasfollowed by 200 μg and 100 μg of anti-PD-1 antibody on days 3 and 4,respectively, as combination therapy. At the indicated timepoints, mice(n=5/group) were injected with CSFE-labeled non-pulsed and SIINFEKLpulsed syngeneic splenocytes. 18 hrs later, splenocytes were harvestedand evaluated for cytolytic activity by measuring the ratio ofCSFE-labeled cells. Immunization with cytokine expressing tumor cellsalone as well as in combination with anti-PD-1 administration resultedin significant CTL activity at 7 days post immunotherapy. By 21 dayspost immunotherapy, however, CTL activity was significantly higher (˜60%lysis) following combination immunotherapy compared to immunization withcytokine expressing tumor cells alone (<20% lysis). Increased CTLactivity was sustained out to 28 days post immunotherapy.

FIG. 1C illustrates the anti-tumor T-cell response induced byadministration of GM-CSF-secreting B16F10 cells alone and in combinationwith an anti-PD-1 antibody, measured as an increase in IFNγ-secretion.On day 0, mice were inoculated subcutaneously with live B16F10 cells. Onday 3, 1×10⁶ irradiated GM-CSF-secreting B16F10 cells (B16.Kd.GM) wereinjected as immunotherapy alone, or immunotherapy was followed by 200 μgand 100 μg of anti-PD-1 antibody on days 3 and 4, respectively, ascombination therapy. On day 17, spleens (n=5/group) were removed and anELISPOT assay was performed to evaluate the number of IFNγ-secretingcells/5×10⁵ splenocytes when stimulated with the B16-specific Kbpeptide, Trp2 (top panel); or irradiated B16F10 (F10; bottom panel). Thenumber of IFNγ-secreting cells was significantly higher in splenocytesfrom mice receiving combination immunotherapy compared to thosereceiving GM-CSF expressing tumor cell immunotherapy alone (B16.Kd.GM)following stimulation either trp2 or irradiated B16F10 cells.

Taken together, these results demonstrate that anti-PD-1 antibodyaugments the anti-tumor T-cell response induced by GM-CSF expressingtumor cell immunotherapy, and support the utility of GM-CSF-secretingtumor cell and anti-PD-1 antibody combination immunotherapy for theinduction and/or enhancement of anti-tumor immunity.

Example 2 Pro-Inflammatory Cytokine Secretion Following GVAX/Anti-PD-1Combination Immunotherapy

A further suggestion as to the potential utility of the combination ofGM-CSF-secreting immunotherapies and anti-PD-1 antibody in eliciting ananti-tumor immune response is the production of pro-inflammatorycytokines such as TNF-alpha, IFN-gamma, IL-2, IL-5, IL-6, IL-10 andMCP-1 upon restimulation in vitro. Release of such cytokines is oftenused as a surrogate marker for monitoring tumor-specific immuneresponses following immunotherapeutic strategies designed to induceanti-tumor immunity.

FIG. 2 illustrates the secretion of pro-inflammatory cytokines followingadministration of GM-CSF-secreting B16F10 cells alone and in combinationwith an anti-PD-1 antibody. On day 0, mice were inoculatedsubcutaneously with live B16F10 cells. On day 3, mice were immunizedwith 1×10⁶ irradiated GM-CSF-secreting B16F10 cells (B16.Kd.GM) asimmunotherapy alone, or immunotherapy was followed by 200 μg and 100 μgof anti-PD-1 antibody on days 3 and 4, respectively, as combinationtherapy. On day 17, splenocytes from selected mice (n=5/group) werecultured with irradiated B16 cells for 48 hrs in 96 well plates.Supernatants were evaluated for secretion levels of the followingcytokines: (A) tumor necrosis factor-alpha (TNFα), (B) interferon-gamma(IFNγ), (C) interleukin-5 (IL-5), (D) interleukin-6 (IL-6), (E)interleukin-10 (IL-10), and (F) monocyte chemotactic protein (MCP)-1.

For each cytokine assayed, cytokine secretion was significantly higherin splenocytes from mice receiving combination immunotherapy compared tothose receiving GM-CSF expressing tumor cell immunotherapy alone. Thus,these results further support the utility of GM-CSF-secreting tumor cellimmunotherapy and anti-PD-1 antibody combination immunotherapy for theinduction and/or enhancement of anti-tumor immunity.

Example 3 Recruitment of T-Cells into Tumors Following GVAX/Anti-PD-1Combination Immunotherapy

FIG. 3 illustrates effector CD8 T-cell infiltration into tumors in micetreated with GM-CSF-secreting B16F10 cells alone or in combination withan anti-PD-1 antibody. High numbers of tumor infiltrating lymphocytes(TIL) within tumors have been shown to correlate with overalltherapeutic benefit (Dunn et al., Nat Immunol. 3(11):991-8., 39 (2002);Smyth et al., Nat Immunol. 2(4):293-9 (2001). Thus, the extent ofinfiltrating lymphocytes in tumors of B16.Kd.GM-treated animals comparedto animals treated with the combination therapy was examined. On day 0,mice were inoculated subcutaneously with live B16F10 cells. On day 3,1×10⁶ irradiated GM-CSF-secreting B16F10 cells (B16.Kd.GM) were injectedas immunotherapy alone, or with 200 μg and 100 μg of anti-PD-1 antibodyon days 3 and 4, respectively, as combination therapy. At the indicatedtimepoints, tumors from mice were excised, digested and stained, andsingle cell suspensions were evaluated by flow cytometry. FIG. 3 showsthe percentage of IFNγ and CD107α expressing cells in the CD8 tumorinfiltrating lymphocyte (TIL) subpopulation from mice immunized with (A)GM-CSF expressing tumor cell immunotherapy alone, and GM-CSF expressingtumor cell immunotherapy alone plus anti-PD-1 antibody. Also shown isthe ratio of CD8+/FoxP3+ cells in the tumor at 3 wks post cellularimmunotherapy (B). Also shown are the kinetics of (C) CD4⁺ T-cells, (D)CD8⁺ T-cells, (E) CD8⁺ and CD107α⁺ T-cells, and (F) CD8⁺ and IFNγ⁺T-cells per 1×10⁶ tumor cells. Tumor progression for these animals isshown in (G).

CD4/IFN-γ co-staining was used to identify effector CD4 T-cells andCD8/IFN-γ or CD8/CD107a co-staining was used to identify effector CD8T-cells within tumors. IFN-γ secreted by T-cells has previously beenshown to correlate with immunoregulatory and anti-tumor properties(Schroder et al., J. Leukoc. Biol. 75(2):163-89 (2004), while expressionof CD107a is associated with cytolytic activity of T-cells (Betts etal., J. Immunol. Methods 281(1-2):65-78, (2003); and Rubio et al., NatMed. 9(11):1377-82 (2003)). Animals receiving the combination therapyshowed an increased in the percentage of activated CD8 effector T-cellswithin tumors compared to animals that received B16.Kd.GM monotherapy(FIG. 3A). Furthermore, HBSS control animals had minimal CD4 and CD8T-cell infiltration into tumors, correlating with tumor progression ofthese animals (FIGS. 3C-D and 3G). Comparatively, animals treated withB16.Kd.GM monotherapy displayed gradual infiltration of T-cells into thetumor environment with peak infiltration seen on day 14, whichcorrelated with the delay in tumor growth observed in this group(*p<0.05 HBSS vs. B16.Kd.GM for all time points). Animals treated withthe combination therapy exhibited a rapid and persistent tumorinfiltration of functional CD4 and CD8 T-cells (*p<0.05 B16.Kd.GM vs.combination therapy on day 21) that correlated with its increasedanti-tumor efficacy.

A favorable intratumoral ratio of effector T-cells (Teff) to regulatoryT-cells (Treg) post immunotherapy has been described to correlate withoverall anti-tumor activity (Quezada et al., J Clin Invest.16(7):1935-45 (2006)) and was therefore assessed in tumors of animalstreated with either B16.Kd.GM monotherapy or the combination therapy. Atthe early time points (week 1 and 2 post immunotherapy), theintratumoral ratio of Teff to Tregs was comparable between B16.Kd.GMmonotherapy and the combination therapy treatment groups (data notshown). In contrast, 3 weeks post immunotherapy, the ratio of Teff toTreg was significantly increased in animals that received thecombination therapy when compared to B16.Kd.GM immunotherapy-treatedanimals (FIG. 3B). The correlation between the improved Teff to Tregratio observed in animals treated with the combination therapy and thebetter control of tumor growth in this group again suggests that thepresence of Tregs in tumors blunts CD8 T-cell activity and that cancerimmunotherapies that shift this ratio in favor of the T effector cellsare more effective at providing anti-tumor responses. In summary, thesedata demonstrated a strict correlation between the potency of theanti-tumor T-cell responses measured in the periphery, the number ofeffector T-cells infiltrating into the tumors and the overall control ofdisease progression in animals treated with either B16.Kd.GM monotherapyor in combination with an anti-PD-1 antibody.

Example 4 Efficacy of Cytokine-Expressing Cellular Immunotherapy PlusAnti-PD-1 Antibody

In vivo studies were carried out using allogeneic B16F10 and autologousCT26 tumor models to determine if an anti-PD-1 antibody in combinationwith a cytokine-expressing cellular immunotherapy can enhance anti-tumorefficacy compared to immunotherapy alone.

For studies utilizing the B16F10 model, mice were inoculatedsubcutaneously with 2×10⁵ live tumor cells on day 0. Mice were theninjected with 1×10⁶ irradiated GM-CSF-secreting B16F10 cells (B16.Kd.GM)as immunotherapy alone, or as combination therapy with 200 μg ofanti-PD-1, followed by 100 μg of anti-PD-1 on the following day. Therapywas started at 3 days, 7 days and 11 days following inoculation toassess the effect of delayed administration on efficacy. Mice weremonitored for the formation of palpable tumors twice weekly. Mice wereassessed daily for any obvious abnormality, and if subcutaneous tumorsreached 15-20 mm-diameter in size or started to ulcerate through theskin, animals were euthanized. A Kaplan-Meier survival curve was usedfor evaluation. Survival curves are shown for mice treated withimmunotherapy at 3 days (FIG. 4A), 7 days (FIG. 4B), or 11 days (FIG.4C) post inoculation.

For each dosing regimen, survival was significantly enhanced in micetreated with the combination therapy compared to immunotherapy alone.HBSS-injected control animals had a MST of 26 days and animals treatedwith B16.Kd.GM immunotherapy on day 3 had a MST of 44 days with 10%long-term survival (*p=0.01 HBSS vs. B16.Kd.GM). MST was furtherprolonged to 57 days in animals that were treated with the combinationtherapy on day 3 with 50% long-term survival (*p<0.05B16.Kd.GM+anti-PD-1 vs. B16.Kd.GM) (FIG. 4A). MSTs were 30 days whenB16.Kd.GM immunotherapy was delayed to either day 7 or day 11 post-tumorchallenge and all animals succumbed to tumor burden by day 45-50. Again,enhanced anti-tumor activity was observed in animals that received thecombination therapy initiated either on day 7 or day 11, with 60%(MST=not reached) and 30% (MST=42 days) of animals surviving long-term,respectively (day 7 initiation of therapy *p<0.005 or day 11 initiationof therapy *p=0.01, long-term survival of B16.Kd.GM+anti-PD-1 vs.B16.Kd.GM) (FIGS. 4B and 4C). Furthermore, on day 90, re-challenge ofsurviving mice on day 90 in both immunotherapy alone and combinationtherapy groups with 2.5-times the initial dose of live tumor cellsdemonstrated the presence of a potent B16-specific memory response,suggesting that the induction of memory responses is B16.Kd.GM dependent(FIG. 4D). Thus, the combination of GM-CSF-secreting tumor cellimmunotherapy and anti-PD-1 antibody is potent enough to significantlydelay tumor growth of well established primary tumors and to maintainmemory responses for protection against tumor re-occurrence.

To evaluate the potency of this combination therapy in a second model,the in vivo study was repeated in the autologous immunogenic CT26 coloncarcinoma tumor model in Balb/c animals. In this study, mice wereinoculated subcutaneously with 2×10⁵ live tumor cells on day 0. On day3, mice were then injected with wither 1×10⁶ irradiated GM-CSF-secretingCT26 cells (CT26.GM) as immunotherapy alone or as combination therapywith 200 μg of anti-PD-1, followed by 100 μg of anti-PD-1 on day 4.Anti-PD-1 antibody alone was also administered as a control, at 200 μgon day 3 post challenge followed by 100 μg anti-PD-1 on day 4.

As shown in FIGS. 5A and 5B, anti-PD-1 antibody and CT26.GMmonotherapies both prolonged the survival of tumor bearing animals to 25days compared to HBSS-injected control animals with a MST of 18 days.Consistent with the findings in the B16 model, CT26 tumor-bearinganimals treated with the combination therapy showed a significantsurvival advantage over animals treated with either monotherapy, with90% of animals surviving long-term (*p<0.01 compared to CT26.GM alone).Thus, anti-PD-1 antibody in combination with GM-CSF-secreting tumor cellimmunotherapy significantly prolonged the survival of animals bearingeither a non-immunogenic or an immunogenic tumor when compared to eithermonotherapy.

Example 6 Enhancement of CD8⁺ T-Cells in the Spleen Induced byGVAX/Anti-PD-1 Combination Immunotherapy

FIG. 6 illustrates the enhancement of the percentage of CD8⁺ T-cells inspleen from mice treated with GM-CSF-secreting B16F10 cells alone or incombination with an anti-PD-1 antibody. On day 0, mice were inoculatedsubcutaneously with live B16F10 cells. On day 3, 1×10⁶ irradiatedGM-CSF-secreting B16F10 cells (B16.Kd.GM) were injected as immunotherapyalone, or with 200 μg and 100 μg of anti-PD-1 antibody on days 3 and 4,respectively, as combination therapy. On day 10 following inoculation,spleens from mice were excised, digested and stained, and splenocyteswere evaluated by flow cytometry. Shown are the percentage of T-cellspositive for (A) CD4; (B) CD8; (C) CD11c; and (D) DX5. The percentage ofCD8 positive T-cells are enhanced in B16.Kd.GM+anti-PD1 treated mice.

Example 7 Enhancement of Memory T-Cells in the Spleen Induced byGVAX/Anti-PD-1 Combination Immunotherapy

FIG. 7 illustrates the percentage of memory T-cells in spleen from micetreated with GM-CSF-secreting B16F10 cells alone or in combination withan anti-PD-1 antibody. On day 0, mice were inoculated subcutaneouslywith live B16F10 cells. On day 3, 1×10⁶ irradiated GM-CSF-secretingB16F10 cells (B16.Kd.GM) were injected as immunotherapy alone, or with200 μg and 100 μg of anti-PD-1 antibody on days 3 and 4, respectively,as combination therapy. On day 10 following inoculation, spleens frommice were excised, digested and stained, and splenocytes were evaluatedby flow cytometry. The percentage of memory T-cells was assessed bystaining cells with for surface markers CD69 and Ly6C. Ly6C has beendescribed as a marker for memory CD8+ T cells (Walumas et al., J.Immunol. 155:1973-1883 (1995) while CD69 is a marker for early T-cellactivation. Memory T-cells were designated as the population of cellsthat were Ly6C+/CD69− within CD4 and CD8 positive cells. FIG. 7 showsthe percentage of Ly6C⁺/CD69⁻ cells within (A) the CD4 subpopulation;and (B) the CD8 subpopulation. The percentage of memory T-cells issignificantly enhanced in spleens from mice treated with theB16.Kd.GM+anti-PD1 combination therapy compared to B16.Kd.GMimmunotherapy alone.

Example 8 Reversal of Anergy and Augmentation of Tumor-Specific T-CellsInduced by GVAX/Anti-PD-1 Combination Immunotherapy

On day 0, 1×10⁶ OT-1 transgenic T-cells were adoptively transferred intoC57BL/6 mice. The following day, mice were intravenously injected with500 mg of SIINFEKL peptide to induce anergy in OT-1 cells. Afterestablishing anergy, on day 11, mice were immunized with 1×10⁶irradiated GM-CSF-secreting B16 cells expressing ovalbumin as asurrogate antigen (GM.ova) as immunotherapy alone or immunotherapy wasfollowed by 200 mg and 100 mg of anti-PD-1 antibody on days 11 and 12,respectively, as combination therapy. At indicated timepoints,peripheral blood leukocyte (PBL) from mice were drawn and evaluated forthe percentage of antigen-specific T-cells by tetramer staining.

FIG. 8 illustrates an increase in the percentage of antigen-specificT-cells was induced by immunotherapy with both GM.ova and GM.ova plusanti-PD-1 antibody (16 days following adoptive transfer) following theestablishment of anergy with SIINFEKL peptide. However, the percentageof antigen-specific T-cells was nearly 2-fold higher at day 16 in GM.ovaplus anti-PD-1 antibody treated mice compared to mice treated withGM.ova alone. These data demonstrate that anti-PD-1 reverses anergy andaugments tumor-specific T-cell response induced by GM-CSF tumor cellimmunotherapy.

Example 9 Antigen-Specific T-Cell Expansion Requires the CombinationTherapy in Each Immunotherapy Boost Cycle

To determine the optimal schedule for administration of each componentof the combination therapy to expand antigen-specific T-cellseffectively in a bi-weekly multi-treatment setting, three differentcombination therapy treatment schedules were evaluated.

To first establish an antigen-specific T-cell response, all B16tumor-bearing animals received GM.ova immunotherapy and anti-PD-1antibody at the first therapy cycle. On day 0, 1×10⁶ ovalbumin-specific,transgenic T-cells (OT-1 cells) were adoptively transferred intotumor-bearing C57BL/6 mice that had been inoculated on day −1 with live2×10⁵ F10.ova cells. On day 3, mice were immunized with 1×10⁶ irradiatedGM-CSF-secreting B16 cells expressing ovalbumin (GM.ova) asimmunotherapy alone or GM.ova immunotherapy was followed by 200 μg and100 μg of anti-PD-1 antibody on days 3 and 4, respectively (the firsttherapy cycle). For subsequent therapy cycles, one group of animalsreceived bi-weekly GM-ova and anti-PD-1 antibody, and another groupreceived bi-weekly anti-PD-1 antibody with GM-ova administered withevery other anti-PD-1 treatment (monthly).

At indicated time points, splenocytes from selected animals wereisolated and evaluated for the presence of ovalbumin-specific CD8+T-cells by co-staining the cells with anti-CD8 antibody and the SIINFEKLtetramer. The study showed the expansion and contraction of thepopulation of ovalbumin-specific CD8 T-cells in the spleen after eachtreatment cycle (FIG. 9A) and that ovalbumin-specific CD8 T-cellsexpanded and peaked 7 days after each therapy cycle in animals thatreceived the cellular immunotherapy and the PD-1 blockade concurrently.In animals that received bi-weekly cellular immunotherapy alone, thepeak of ovalbumin-specific CD8 T-cell expansion from the secondimmunotherapy cycle was observed 2 weeks later (day 28). In contrast, inanimals that received bi-weekly anti-PD-1 antibody and monthly GM.ovaimmunotherapy, the expansion of the ovalbumin-specific CD8 T-cells wasonly observed after the treatment cycle that consisted of the cellularimmunotherapy and the PD-1 blockade and not when only the antibody wasadministered.

The effector phenotype of these ovalbumin-specific CD8 T-cells wasconfirmed by co-staining the cells with the activation markers, CD107aand IFNγ (FIGS. 9B and C). In animals that received the combinationtherapy at each therapy cycle, a similar trend in the number oftetramer-positive cells and the number of cells with an effectorphenotype was observed. However, in animals that received bi-weeklycellular immunotherapy alone or bi-weekly anti-PD-1 antibody and monthlyGM.ova immunotherapy, the peak of the IFNγ-secreting ovalbumin-specificCD8 T-cells did not always correlate with the peak of totalovalbumin-specific CD8 T-cells. After the second treatment cycle, in thebi-weekly cellular immunotherapy alone group, the number ofIFNγ-secreting ovalbumin-specific CD8 T-cells peaked on day 21 whiletotal ovalbumin-specific CD8 T-cells peaked on day 28. Similarly, in thebi-weekly anti-PD-1 antibody and monthly GM.ova immunotherapy group, thenumber of IFNγ-secreting ovalbumin-specific CD8 T-cells peaked on day 21while total ovalbumin-specific CD8 T-cells did not change. After thesecond treatment cycle, the data showed that the combination therapymost efficiently expanded the number of IFNγ-secretingovalbumin-specific CD8 T-cells (21.92+/−3.31×106 cells) and thatcellular immunotherapy alone (13.57+/−1.95×106 cells) is more effectivethan anti-PD-1 antibody alone (6.11+/−1.20×106 cells). In summary, thesedata suggested that readministration of the cellular immunotherapy withthe anti-PD-1 antibody in subsequent immunotherapy cycles was requiredto reactivate these T-cell responses.

The present invention is not to be limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention. Indeed, various modifications of the invention inaddition to those described herein will become apparent to those havingskill in the art from the foregoing description and accompanyingdrawings, Such modifications are intended to fall within the scope ofthe appended claims. All references cited herein are hereby incorporatedby reference in their entireties.

What is claimed is:
 1. A method of cancer immunotherapy comprising:administering a combination of a cytokine-expressing cellularimmunotherapy and an antibody that specifically binds to humanProgrammed Death 1 (anti-PD-1) to a subject with cancer, whereinadministration of the combination results in enhanced therapeuticefficacy relative to administration of the cytokine-expressing cellularimmunotherapy or the anti-PD-1 antibody alone.
 2. The method of claim 1,wherein the cytokine-expressing cellular immunotherapy expresses GM-CSF.3. The method of claim 1, wherein the combination comprises cells thatare autologous to the subject.
 4. The method of claim 1, wherein thecombination comprises cells that are allogeneic to the subject.
 5. Themethod of claim 1, wherein the combination comprises bystander cells. 6.The method of claim 1, wherein the cytokine-expressing cellularimmunotherapy is rendered proliferation-incompetent by irradiation. 7.The method of claim 1, wherein the subject is a human.
 8. The method ofclaim 1, wherein the cancer is a prostate cancer.
 9. The method of claim1, wherein the cancer is a non-small cell lung carcinoma.
 10. The methodof claim 1, wherein the cancer is a melanoma.
 11. The method of claim 1,wherein the cancer is a colorectal cancer.
 12. The method of claim 1,wherein the cancer is a renal cell carcinoma.
 13. The method of claim 1,wherein the cancer is an ovarian cancer.
 14. The method of claim 4,wherein the allogeneic cells are a tumor cell line selected from thegroup consisting of a prostate tumor line, a non-small cell lungcarcinoma line and a pancreatic cancer line.
 15. The method of claim 9,wherein the cytokine-expressing cellular immunotherapy is renderedproliferation-incompetent by irradiation.
 16. The method of claim 1,wherein the anti-PD-1 antibody is monoclonal.
 17. The method of claim 1,wherein the anti-PD-1 antibody is mammalian.
 18. The method of claim 1,wherein the anti-PD-1 antibody is fully human.
 19. The method of claim1, wherein the anti-PD-1 antibody is humanized.
 20. The method of claim1, wherein the cytokine-expressing cellular immunotherapy isadministered subcutaneously.
 21. The method of claim 1, wherein thecytokine-expressing cellular immunotherapy is administeredintratumorally.
 22. The method of claim 1, wherein thecytokine-expressing cellular immunotherapy is intradermally.
 23. Themethod of claim 1, wherein the anti-PD-1 antibody is administeredsubcutaneously.
 24. The method of claim 1, wherein the anti-PD-1antibody is administered intratumorally.
 25. The method of claim 1,wherein the anti-PD-1 antibody is administered intradermally.
 26. Themethod of claim 1, wherein the anti-PD-1 antibody is administeredintravenously.
 27. The method of claim 1, wherein the anti-PD-1 antibodyis administered simultaneously with administration of theGM-CSF-expressing cellular immunotherapy.
 28. The method of claim 1,wherein the anti-PD-1 antibody is administered one, two, three, four,five, six, seven, or more days after administration of theGM-CSF-expressing cellular immunotherapy.
 29. The method of claim 1,wherein the GM-CSF-expressing cellular immunotherapy is administeredone, two, three, four, five, six, seven, or more days afteradministration of the anti-PD-1 antibody.
 30. The method of claim 1,wherein the anti-PD-1 antibody is administered simultaneously withadministration of the GM-CSF-expressing cellular immunotherapy on abiweekly basis.
 31. The method of claim 1, wherein enhanced therapeuticefficacy is measured by increased overall survival time.
 32. The methodof claim 1, wherein enhanced therapeutic efficacy is measured byincreased progression-free survival.
 33. The method of claim 1, whereinenhanced therapeutic efficacy is measured by decreased tumor size.
 34. Acomposition for cancer therapy comprising: a cytokine-expressingcellular immunotherapy and an antibody that specifically binds to humanProgrammed Death 1 (anti-PD-1), wherein administration of thecomposition to a subject with cancer results in enhanced therapeuticefficacy relative to administration of the cytokine-expressing cellularimmunotherapy or the anti-PD-1 antibody alone.
 35. The composition ofclaim 34, wherein the cells of said cytokine-expressing cellularimmunotherapy are autologous to the subject.
 36. The composition ofclaim 34, wherein the cells of said cytokine-expressing cellularimmunotherapy are allogeneic to the subject.
 37. The composition ofclaim 34, wherein the cells of said cytokine-expressing cellularimmunotherapy cells are bystander cells.
 38. The composition of claim34, wherein the cells of said cytokine-expressing cellular immunotherapyare rendered proliferation-incompetent by irradiation.
 39. Thecomposition of claim 36, wherein said allogeneic cells are a tumor cellline selected from the group consisting of a prostate tumor line, anon-small cell lung carcinoma line and a pancreatic cancer line.
 40. Thecomposition of claim 34, wherein the anti-PD-1 antibody is monoclonal.41. The composition of claim 34, wherein the anti-PD-1 antibody ismammalian.
 42. The composition of claim 34, wherein the anti-PD-1antibody is fully human.
 43. The composition of claim 34, wherein theanti-PD-1 antibody is humanized.
 44. The composition of claim 34,wherein enhanced therapeutic efficacy is measured by increased overallsurvival time.
 45. The composition of claim 34, wherein enhancedtherapeutic efficacy is measured by increased progression-free survival.46. The composition of claim 34, wherein enhanced therapeutic efficacyis measured by decreased tumor size.
 47. The composition of claim 34,wherein the cytokine-expressing cellular immunotherapy expresses GM-CSF.48. A kit comprising: a first composition comprising acytokine-expressing cellular immunotherapy and a second compositioncomprising an antibody that specifically binds to human Programmed Death1 (PD-1), wherein administration of the first and second compositions toa subject with cancer results in enhanced therapeutic efficacy relativeto administration of the cytokine-expressing cellular immunotherapy orthe antibody that specifically binds to PD-1 alone.
 49. The kit of claim48, further comprising instructions directing administration of thefirst and second compositions.
 50. The kit of claim 48, wherein saidinstructions direct simultaneous administration of the first and secondcompositions.
 51. The kit of claim 48, wherein said instructions directadministration of the first and second compositions simultaneously. 52.The kit of claim 48, wherein said instructions further directadministration of the first and second compositions simultaneously on abiweekly basis.
 53. The kit of claim 48, wherein said instructionsdirect administration of the second composition one, two, three, four,five, six, seven, or more days subsequent to administration of the firstcomposition.
 54. The kit of claim 48, wherein said instructions directadministration of the first composition one, two, three, four, five,six, seven, or more days subsequent to administration of the secondcomposition.
 55. The kit of claim 48, wherein the cytokine-expressingcellular immunotherapy expresses GM-CSF.